EP1603586A1 - 14-3-3 protein for prevention and treatment of fibroproliferative disorders - Google Patents

Abstract

The present invention relates generally to methods for the prevention and/or treatment of fibroproliferative disorders in a mammal. More particularly, this invention relates to 14-3-3 proteins which mediate collagenase expression and content of the tissues and organs of a mammal. It also relates to a method of increasing the collagen content of the skip of a mammal. Furthermore it relates to the prevention, alleviation and/or treatment of skip having a low collagen content. The invention also relates to a method of screening for compounds that are capable of increasing the collagen content of the skip, and to compounds identified by said method. The invention also discloses combinations of a weak acid proteins with 14-3-3 that provides complimentary fibroproliferative therapy.

Description

-3-3 PROTEIN FOR PREVENTION AND TREATMENT OF FIBROPROLIFERATIVE DISORDERS

FIELD OF THE INVENTION

5 The present invention relates generally to methods for the prevention and/or treatment of fibroproliferative disorders in a mammal. More particularly, this invention relates to proteins which mediate collagenase expression and content of the tissues and organs of a mammal. It also relates to a method of increasing the collagen content of

10 the skin of a mammal. Furthermore it relates to a method for the prevention, alleviation and/or treatment of skin having a low collagen content. The invention also relates to a method of screening for compounds that are capable of increasing the collagen content of the skin, and to compounds identified by said method. The invention also discloses

15 combinations of proteins with other pharmaceutically active substances. BRIEF DESCRIPTION OF THE PRIOR ART

The human body responds to trauma and injury by the formation of scars. When the normal wound healing response is malfunctioning wounds will heal with formation of excessive scars. Fibrosis, which is a

20 disorder belonging to the group of fibroproliferative disorders is characterized by excessive scarring and is attributed to a continuation of the wound healing response which causes an excessive production and deposition of collagen. Patients that may benefit from an efficient anti- fibrotic therapy number in the millions. There is, however, no effective

25 treatment of such fibroproliferative disorders available. Fibroproliferative disorders are believed to be caused by trauma, surgery, infection environmental pollutants, alcohol and other types of toxins. There are various examples of fibroproliferative disorders, including the formation of scar tissue after a heart attack, which scar tissue impairs the ability of the

30 heart to pump. Diabetes frequently causes damage and scarring in the kidneys which leads to a progressive loss of kidney function. Even after surgery, scar tissue can form between internal organs causing contracture, pain and malfunctioning. Fibroproliferative disorders can be acute or chronic, but they always share a common characteristic of excessive collagen accumulation and an associated loss of function when normal tissue is replaced with scar tissue. As far as the acute form of fibrosis is concern, it occurs as a common response to various forms of trauma including burns, surgery severe injuries, infections and radiation treatment. As far as the chronic form is concerned this is believed to be caused by diabetes, hypertension, various viral infections like viral hepatitis which induce a progressive fibrosis which causes a continuous loss of tissue ^• function. Typically, as far as internal organs are concerned liver, kidney and lung are most affected.

For both, acute and chronic forms of fibrosis, there is currently no effective treatment available.

The skin has a protective role and provides a barrier between our organs and tissues, giving support, balancing fluid metabolism, regulating temperature etc. The skin's functions are manifold, and therefore an intact integument is necessary so that these functions can be fulfilled. Each year more than two million burn injuries demand medical care, and approximately three quarters of a million people suffer every year from diabetic leg ulcers, thus creating a large market for replacement skin grafts.

Summarized briefly, the skin comprises dermal and epidermal layers each of them being derived from different stem cells, with the former being formed by fibroblasts and the latter being a product of the differentiation of keratinocytes. In natural skin, fibroblasts produce collagen and elastin that serve as a foundation upon which keratinocytes differentiate into the epidermal layer. The epidermis is the thin outer layer of the skin, which itself consists of the following three parts; the stratum corneum which consists of fully mature keratinocytes which contain fibrous proteins (keratins). The outermost layer is continuously shed. The stratum corneum prevents the entry of most foreign substances as well as the loss of fluid from the body. Secondly, beneath the stratum corneum there lies the layer of keratinocytes (squamous cells), which, as the name implies, contains living keratinocytes (squamous cells), which mature and form the stratum corneum. Thirdly, the deepest layer of the epidermis is the basal layer which contains basal cells. Basal cells continually divide, forming new keratinocytes, replacing the old ones that are shed from the skin's surface. In addition to the aforementioned three layers the epidermis also contains melanocytes which are usually evenly dispersed and which are cells that produce melanin. Below the epidermis there is the dermis which is the middle layer of the skin. It contains blood vessels, fibroblasts, hair follicles, sweat glands, lymph vessels, collagen bundles and nerves. The dermis is held together by collagen which in turn is produced by fibroblasts. The dermis also contains pain and contact receptors. Below the dermis there is the subcutis which is the deepest layer of the skin. The subcutis consists of a network of collagen and fat cells and it helps to conserve the body's heat. Furthermore it protects the body from injury by acting as a "shock absorber". In a, human population there are both genetic and cultural differences as to the thickness of the subcutis layer.

When skin wounds heal, complex processes are initiated which change temporally and spatially. Typical phases of wound healing are granulation, inflammation, wound re-epitheliasalion via migration and proliferation of keratinocytes and matrix remodeling. The latter process involves major reorganization of molecules of the extracellular matrix including the prominent collagens. Eventually a scar is formed. Often, excess deposition of collagen and other extracellular matrix proteins (ECM) leads to hypertrophic scars, or even keloids, which are scars increasing in area and spreading into the surrounding healthy skin. Keloids are dermal fibrotic lesions that are a variation of the normal wound healing process. They usually occur during the healing of a deep skin wound. Both hypertrophic scars and keloids are included along a spectrum of the above mentioned fibroproliferative disorders. These abnormal scars appear to result from the loss of the control mechanisms that normally regulate the fine balance of tissue repair and regeneration. The excessive proliferation of normal tissue healing processes results in both hypertrophic scars and keloids. The production of extracellular matrix proteins, collagen, elastin and proteoglycanes is believed to be due to a prolonged inflammatory process in the wound. Hypertrophic scars are raised, erythematous, fibrotic lesions, that usually remain confined within the borders of the original wound. These scars occur within months of the initial trauma and have a tendency to remain stable or regressive with time. Keloid formation, however, can occur within a year after injury, and keloids enlarge well beyond the original scar margin. The most frequently involved sites of keloids are areas of the body that constantly are subjected to high skin tension. Wounds on the anterior chest, shoulders, flexor surfaces of the extremities, anterior neck and wounds that cross skin tension lines are more susceptible to abnormal scar formation.

One factor that seems to play a role in the development of abnormal scars is time, that is if the healing time is greater than three weeks there is a pronounced likelihood that abnormal scar formation will occur. Furthermore wounds that have been subjected to a prolonged inflammation, whether due to a foreign body, infection, burns or inadequate wound closure, are also at risk of abnormal scar formation. Areas of chronic inflammation such as an earring site or a site of repeated trauma are also more likely to develop keloids. Some evidence supports a relationship between genetic predisposition and an individual's propensity to form keloid scars. Genetic associations have been found between human leukocyte antigen (HLA)-B14, HLA-B21 , HLA-BW16, HLA-BW35, HLA-DR5, HLA-DQW3, blood group A and the development of abnormal scars. No consistent pattern yet exists in the mode of genetic transmission, which is reported as occurring in both an autosomal dominant and autosomal recessive pattern.

After the initial insult to the skin and the formation of a wound clot, the balance between granulation tissue degradation and biosynthesis becomes essential to adequate healing. Extensive studies of the biochemical and cellular composition of keloids compared to mature scar tissue demonstrate significant differences. Keloids have an increased density of blood vessels, higher mesenchymal, thickened epidermal layer, and an increase in mucinous ground substance. The alpha-smooth muscle actin fibroblasts, myofibroblasts, important for contractal situations, are few if present at all. The collagen fibrils in keloids are more regular, abnormally thick and have unidirectional fibers arranged in a highly stressed orientation. Biochemical differences in collagen content in normal hypertrophic scars and keloids have been examined in numerous studies. For example collagen synthesis in keloids is three times greater than in hypertrophic scars and believed to be twenty times greater than in normal scars. Type III collagen, chondroitin-4-sulfate and glycosaminoglycan contents is higher in keloids than in both hypertrophic and normal scars. Collagen cross linking is greater in normal scars, while keloids have immature cross links that do not form normal scar stability. The increased number of fibroblasts, which are recruited to the site of tissue damage, synthesize an overabundance of fibronectin, and receptor expression is increased in keloids. Mast cell population within keloid scars is also increased and subsequently an increase in histamin production occurs. Growth factors and cytokines are involved intimately in a cycle of wound healing. IFN-alpha, IFN-beta and IFN-gamma reduce fibroblasts synthesis of collagen types I, III and possibly VI. A relationship between irnmunoglobulines and keloid formation appears to exist, while levels of immunoglobulin IgG and IgM are normal in a serum of patients with keloids, the concentration of IgG in the scar tissues elevated in comparison to hypertrophic and normal scar tissue.

Scars contain few cells and blood vessels, but high amounts of connective tissue. Scars not only present a severe cosmetic problem, but also affect the function of the skin: the elasticity of scars is greatly reduced compared to healthy skin due to the minor quality of collagen fibers; also appendages like sweat glands, hair follicles and melanocytes are missing. Therefore the scarred skin cannot fulfill its normal function as a barrier for both fluid metabolism and heat loss. This leads to severe problems of the affected individuals when the affected skin is exposed for example to mechanical stress or extreme temperatures. Currently, no therapy exists which reliably prevents or treats hypertrophic scar formation and keloids. Usually scars are treated mechanically by excision or by laser or cooling techniques; however these techniques again lead to wounds and eventually scars. Also collagen or hyaluronic acid may be injected into scar tissue to make the scar surface more even. However, the material is reabsorbed within months, and the treatment has to be repeated every 6- 12 months. Also this is mainly a cosmetic treatment ameliorating scar appearance. No single therapy is rated experimentally to be the most effective form of treating keloid scars. In the past, keloids have been treated by occlusive dressings, including silicone gel sheets and silicone occlusive dressings which can be worn for as long as 24 hours per day for up to one year. The silicone does not appear to enter the skin, and the antikeloid effect appears to be secondary to both occlusion and hydration. Furthermore keloids have been treated by mechanical compression dressings, in particular in relation to ear lobe keloids. They are usually custom made for the patient and are most effective if worn 24 hours per day. The mechanism of action is unknown, but is believed to be partially due to a reduction of oxygen tension in the wound through occlusion of small vessels. Furthermore keloids have been treated by intralesional steroid injections which apparently act by diminishing collagen synthesis, decreasing mucinous ground substance. There are the usual adverse side effects of prolonged treatment with steroids, including atrophy of the skin or subcutaneous tissue, hypopigmentation, necrosis, ulceration, etc. Keloids have also been treated by additional excision surgery which is usually combined with other kinds of therapy like external radiation, steroid injection and/or pressure therapy. Cryosurgery has also been applied, using liquid nitrogen to cause cell damage and to affect the microvasculature which causes subsequent stasis, thrombosis and transudation of fluid that result in cells anoxia. Quite recently keloids have also been treated by using intralesional injections of IFN-alpha, IFN-beta and IFN-gamma. Some studies have demonstrated that these interferons reduce fibroblast synthesis of collagen types I, III and possibly VI, but the exact mechanism remains yet to be elucidated.

None of the above mentioned forms of therapy has proved to be successful in the treatment of keloids. With respect to fibroproliferative disorders in general, there is currently no single therapy available that can be considered as particularly effective, and current therapy is merely symptomatic.

Stratifin, which is also called 14-3-3 sigma, is a 28kDa protein which is known to be involved in cell cycle progression and cell cycle control as an intracellular protein (see for example Prasad et al., 1992, Cell Growth

Differ., 3:507-513). This is also the role that is attributed to 14-3-3-proteins in general (van Hemert et al., 2001, Bioessays, 23: 936-946).

US 6,335,156 discloses that 14-3-3-sigma protein is known to have a growth suppressing effect on malign tumor cells. Katz and Taichman, 1999, J. Invest. Dermatol., 112: 818-821 , report on the secretion of 14-3-3 sigma protein by epidermal keratinocytes. Dellambra et al., 1995, J. Cell Sci., 108: 3569-3579, report that 14-

3-3 sigma is involved in a signal transduction pathway which is involved in the terminal differentiation of keratinocytes. Reichelt and Magin (2002, J. Cell Sci., 115: 2639-2650) report that a hyperproliferation of the epidermis can be observed which is accompanied by the induction of different genes, including the gene for 14-

3-3sigma. No specific function for 14-3-3 sigma is mentioned other than its ability to induce a G2 block of cells. No mention is made in the prior art with respect to the utility of stratifin as a compound to be used in connection with fibroproliferative disorders.

Accordingly, there remains a need for an effective treatment of fibroproliferative disorders. Furthermore, there remains a need for an effective treatment of hypertrophic scarring and keloids. There is a need for a method that allows the identification of compounds that are useful as collagen-increasing agents, cosmetics and/or anti-wrinkle-compounds.

Furthermore, there remains a need for a method for increasing the collagen of the skin. SUMMARY OF THE INVENTION

The present invention reduces the difficulties and disadvantages of the prior art by providing a method for the prevention and/or treatment of fibroproliferative disorders in a mammal, comprising administering an effective amount of a 14-3-3 protein, an active fragment thereof, or a functional analogue thereof; or of a nucleic acid encoding for said protein in order to increase collagenase. The present invention also provides a method for increasing the collagen content in the skin by reducing the collagenase content in a tissue or organ.

Therefore, in a first aspect of the present invention there is provided a method for the prevention and/or treatment of a fibroproliferative disorder in a mammal, comprising administering an effective amount of a 14-3-3 protein, any active 14-3-3 fragment thereof, or a functional analogue thereof, or of a nucleic acid encoding for said protein to said mammal. In a second aspect of the present invention, there is provided a method of increasing collagen content of the skin and/or for the prevention, alleviation and/or treatment of skin having a low collagen content, comprising administering an antibody or binding fragment thereof, wherein the antibody or fragment specifically binds to a 14-3-3 protein, its receptors, or its fragments, or administering a nucleic acid that is antisense to a nucleic acid encoding for said mammalian 14-3-3 protein or administering siRNA directed towards a nucleic acid encoding for a 14-3- 3- protein. In a third aspect of the present invention, there is provided a method of screening for compounds that are capable of increasing the collagen content of the skin, comprising:

- providing cells expressing or expressing and secreting a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID NO: 1 or 2, or an active fragment thereof, or a functional analogue thereof,

- incubating said cells with a potentially active compound,

- measuring the expression and/or secretion of said protein, or

- providing a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID NO: 1 or

2, or an active fragment thereof, or a functional analogue thereof, - incubating said protein with cells expressing collagenase, together with a potentially active compound,

- measuring the expression of collagenase, an active compound being a compound that reduces/inhibits expression or secretion of said protein, or a compound that reduces/inhibits expression of collagenase. In a fourth aspect, there is provided use of a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein in the treatment of a fibroproliferative disorder in a mammal.

In a fifth aspect, there is provided use of an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, its fragments, or its receptors, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein to increase the collagen content of a mammal's skin.

In a sixth aspect of the present invention, there is provided use of a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein in the manufacture of a medicament for the treatment of fibroproliferative disorders.

In a seventh aspect of the present invention, there is provided use of an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein, in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.

In an eighth aspect of the present invention, there is provided a compound identified by a method according to the present invention. In yet another aspect, the invention provides the use of a mixture of a pharmaceutically acceptable weak acid or a derivative thereof, an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein in the manufacture of a medicament for the treatment of fibroproliferative disorder.

In another form, the invention provides a pharmaceutically acceptable weak acid or a derivative thereof, an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein together with a pharmaceutically acceptable carrier therefor. Derivatives of the weak acids may encompass any pharmaceutically acceptable ester or salt or other suitable derivative. In a more particular form of the invention, the ratio of a pharmaceutically acceptable weak acid or a derivative thereof, an example of which is acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein may range from 1 :1 to 1 :1000 on a dry weight basis mixed in cream or solution. A mixture in a 1 :1 ratio has been found to be effective when the weak acid is acetylsalicylic acid.

As is described in the detailed description, the mixture provides complimentary benefits for the proposed treatment since it not only suppresses the expression of collagen, but also significantly increases the expression of collagenase.

It is also envisaged that the 14-3-3 protein, any active fragment thereof, or a functional analogue thereof; or a nucleic acid encoding for a 14-3-3 protein may be used in implantable medical devices, such as, for example stents used in angioplasty or for other therapeutic purposes to prevent fibroproliferative disorders in patients. Combinations with the weak acids mentioned above are also envisaged for such purposes. The protein, etc. may be coated onto the implant or otherwise incorporated into it in a suitable manner as would be apparent to a person skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally described the invention, reference with now be made to the accompanying drawings, showing by way of illustration preferred embodiments thereof. Fig. 1 illustrates the expression of collagenase by fibroblasts co- cultured with human dermal keratinocyles.

Fig. 2 illustrates the expression of collagenase mRNA and 18S ribosomal RNA by dermal fibroblasts which received various percentages of keratinocyte conditioned medium.

Fig. 3 illustrates the expression of collagenase and 18 S ribosomal RNA by dermal fibroblasts which have received keratinocyte condition medium from keratinocytes grown in test medium at the indicated time intervals. Fig. 4 illustrates the results of an activity test of protein that is present in keratinocyte conditioned medium and that has been precipitated by ammonium sulfate.

Fig. 5 illustrates a section of a dermal/epidermal skin substitute stained with H Si E (panel A); panel B shows the expression of collagenase and 18 S ribosomal RNA from fibroblasts in a collagen matrix, keratinocytes in the epidermal layer and fibroblasts alone on plastic.

Fig. 6 illustrates collagenase activity in keratinocyte conditioned medium.

Fig. 7 shows an SDS-PAGE gel showing the purification of stratifin. Fig. 8 illustrates the effects of various doses of stratifin on the collagenase mRNA expression in fibroblasts.

Fig. 9 illustrates that the KDAF (Stratifin) collagenase stimulatory effect is not permanent.

Fig. 10 illustrates that Keratinocyte conditioned medium contains a collagenase stimulating factor that increases the expression of collagenase in a time dependent fashion.

Fig. 11 illustrates that that α/β form of protein 14-3-3 has also collagenase stimulatory effect in fibroblasts.

Fig. 12 illustrates collagenase stimulatory effects of RNA by Northern Analysis.

Fig. 13A illustrates an analysis of KDAF protein fragments on native gel (upper panel) and an analysis of KDAF protein fragments on SDS-Page (lower panel). Fig. 13B illustrates that deletion of up to 75 amino acids is not critical to the KDAF collagenase stimulatory effect.

Figs. 14A and B illustrate Northern Analysis using cDNA probe for collagenase (top panel) or 185 ribosomal RNA (bottom panel). Fig. 15 illustrates evaluation of proteins if fractions were run in a

SDS-PAGE using an un-fractionated Fetal Bovine Serum sample as a control (Panel A) or used to treat dermal fibroblast to evaluate its collagenase activity (Panel B). Panel C demonstrates the pattern of Western blot analysis showing the presence and quantities of KDAF. Fig. 16 illustrates SDS-PAGE followed by Western blotting (upper panel) that human serum also contains KDAF.

Fig. 17 illustrates the histological appearance of KDAF in treated and untreated healing wounds in rats.

Fig. 18 illustrates how the combination of KDAF and acetylsalicylic acid suppresses the expression of collagen and increases the expression of collagenase.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains but should not be interpreted as limiting the scope of the present invention.

As used herein, the term "14-3-3 protein" is meant to designate any protein belonging to the family of 14-3-3 proteins. These proteins in general have a core region encoding an amphipathic groove that binds a multitude of client proteins that have conserved 14-3-3 recognition sequences. The amino and carboxy termini of 14-3-3 proteins are much more diverse than the core region and may interact with isoform-specific client proteins and/or confer specialized sub-cellular and tissue localisation. 14-3-3 proteins have so far only been found in eukaryotes, in different isoforms including, beta/alpha, gamma, epsilon, eta, sigma, tau/theta and zeta/delta (Yaffe MB, FEBS Letters 513: 53-57, 2002). They also have been found in yeasts, Drosophila and plants (Martens GJM, Piosik PA, Danen EHJ, Evolutionary Conservation of the 14-3-3 protein, Biochem. Biophy. Res. Comm. 184:1456-1459, 1992).

As used herein the term "functional analogue" of a protein is meant to designate a protein that carries out the same essential function as a 14- 3-3 protein without necessarily having the same sequence. This includes active fragments and variants of a 14-3-3 protein. Variants include deletion and/or substitution of at least one amino acid of the protein, e.g., by manipulation of the nucleic acid encoding the protein or by altering the protein itself. An active fragment or portion of the protein means a stretch of amino acid residues of sufficient length or having amino acids deleted therein, while retaining its essential function. Specific Embodiments

The present invention provides methods for the prevention and/or treatment of fibroproliferative disorders in a mammal. More particularly, this invention provides proteins, nucleic acids, and antibodies, which mediate collagen expression and content of the tissues and organs of a mammal. It also relates to a method of increasing the collagen content of the skin of a mammal. Furthermore it provides a method for the prevention, alleviation and/or treatment of skin having a low collagen content, such as those of non-healing wounds. The invention also provides a method of screening for compounds that are capable of increasing the collagen content of the skin, and to compounds identified by said method.

It has surprisingly been found that the 14-3-3 protein, stratifin, which can also be referred to as "KDAF" or "Keratinocyte Derived Anti-fibrogenic Factor", can induce collagenase expression in fibroblasts when added to the surrounding medium, which leads to the degradation of excess collagen in scars and therefore to the prevention of scars when applied during or before scar formation. Furthermore it allows the treatment of already existing hypertrophic scars. Stratifin is naturally released from human skin cells and its potency is more than 20 fold higher than a well known anti-fibrogenic cytokine, such as IFN-alpha2b. Stratifin is thus an anti-fibrogenic factor for treatment of fibroproliferative disorders, especially fibrosis disorders, e.g. fibrosis of liver, kidney, lung, bowel, heart, pancreas, peritoneum, skin or other organs, preferably of the skin. Fibrosis in general can lead to cirrhosis, idiopathic pulmonary fibrosis, glomerulosclerosis and hypertrophic scars.

KDAF is a natural Ketatinocyte releasable protein whose collagenase stimulating effects on dermal fibroblasts has been confirmed in a dose and time dependent fashion, and as such is believed to function as a wound healing stopping signal after wound re-epithelealgation.

Therefore, in accordance with a first aspect of the present invention there is provided a method for the prevention and/or treatment of a fibroproliferative disorder in a mammal, comprising administering an effective amount of a 14-3-3 protein or a functional analogue thereof, or an active fragment thereof, or a nucleic acid encoding for such a protein. Use of a 14-3-3 protein or a functional analogue thereof, or an active fragment thereof, or a nucleic acid encoding for such a protein in the treatment of a fibroproliferative disorder in a mammal is likewise provided.

In one embodiment, the 14-3-3 protein is a mammalian protein, preferably of human origin. In another preferred embodiment, the protein is an isoforrn of 14-3-3 sigma protein, including (see page 12, lines 18 and 19) isoforms found in human, as well as other forms found in plants, yeast and fungi as a monomer, homodimer, heterodimer. In another embodiment, the protein is any other form that can function as a collagenase stimulating factor as can be determined by methods as set out in the examples below. In another embodiment, the protein comprises a fragment of a 14-3-3 protein, or an isoforrn thereof, that functions as a collagenase stimulating factor as can be determined by methods as set out in the examples below. Such proteins and fragments can be naturally or synthetically produced using conventional methods well known to those skilled in the art. Synthetic production of the proteins or fragments thereof can provide an inexpensive, safe and simple method of producing the desired protein or fragment thereof . Fragments may be administered via typical routes, including topically and systemically.

In one embodiment, the 14-3-3 protein is a sigma 14-3-3 protein having a sequence as represented by SEQ ID NO: 1 as follows: SEQ ID NO: 1

1 merasliqka klaeqaerye dmaafmkgav ekgeelscee mllsvaykn vvggqraawr 61 vlssieqksn eegseekgpe vreyrekvet elqgvcdtvl glldshlike agdaesrvfy

121 Ikmkgdyyry laevatgddk kriidsarsa yqeamdiskk empptnpirl glalnfsvfh

181 , yeianspeea islakttfde amadl tlse dsykdstlim qllrdnltlw tadnageegg

241 eapqepqs

Preferably, the mammalian 14-3-3 protein is an α/β 14-3-3 protein having a sequence as represented by SEQ ID No: 2 as follows:

SEQ ID NO: 2

1 mtmdkselvq kaklaeqaer yddmaaamka vteqghelsn eernllsvay knvvgarrss

61 wrvissieqk ternekkqqm gkeyrekiea elqdicndvl elldkylipn atqpeskvfy

121 Ikmkgdyfry Isevasgdnk qttvsnsqqa yqeafeiskk emqpt pirl glalnfsvfy

181 yeilnspeka cslaktafde aiaeldtlne esykdstlim qllrdnltlw tsenqgdegd

241 agegen

In another embodiment, the nucleic acid encoding for said sigma 14-3-3 protein has a sequence as represented by SEQ ID NO: 3 as follows:

Seq. ID NO. 3: 14-3-3 Sigma

ORIGIN

1 gagcaggaga gacacagagt ccggcattgg tcccaggcag cagttagccc gccgcccgcc

61 tgtgtgtccc cagagccatg gagagagcca gtctgatcca gaaggccaag ctggcagagc

121 aggccgaacg ctatgaggac atggcagcct tcatgaaagg cgccgtggag aagggcgagg 181 agctctcctg cgaagagcga aacctgctct cagtagccta taagaacgtg gtgggcggcc

241 agagggctgc ctggagggtg ctgtccagta ttgagcagaa aagcaacgag gagggctcgg

301 aggagaaggg gcccgaggtg cgtgagtacc gggagaaggt ggagactgag ctccagggcg

361 tgtgcgacac cgtgctgggc ctgctggaca gccacctcat caaggaggcc ggggacgccg

421 agagccgggt cttctacctg aagatgaagg gtgactacta ccgctacctg gccgaggtgg 481 ccaccggtga cgacaagaag cgcatcattg actcagcccg gtcagcctac caggaggcca

541 tggacatcag caagaaggag atgccgccca ccaaccccat ccgcctgggc ctggccctga

601 acttttccgt cttccactac gagatcgcca acagccccga ggaggccatc tctctggcca

661 agaccacttt cgacgaggcc atggctgatc tgcacaccct cagcgaggac tcctacaaag

721 acagcaccct catcatgcag ctgctgcgag acaacctgac actgtggacg gccgacaacg 781 ccggggaaga ggggggcgag gctccccagg agccccagag ctgagtgttg cccgccaccg

841 ccccgccctg ccccctccag tcccccaccc tgccgagagg actagtatgg ggtgggaggc

901 cccacccttc tcccctaggc gctgttcttg ctccaaaggg ctccgtggag agggactggc

961 agagctgagg ccacctgggg ctggggatcc cactcttctt gcagctgttg agcgcaccta

1021 accactggtc atgcccccac ccctgctctc cgcacccgct tcctcccgac cccaggacca 1081 ggctacttct cccctcctct tgcctccctc ctgcccctgc tgcctctgat cgtaggaatt

1141 gaggagtgtc ccgccttgtg gctgagaact ggacagtggc aggggctgga gatgggtgtg

1201 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg cgcgcgcgcc agtgcaagac cgagattgag

1261 ggaaagcatg tctgctgggt gtgaccatgt ttcctctcaa taaagttccc ctgtgacact

1321 ln another embodiment, the nucleic acid encoding for said 14-3-3 alpha/beta protein has a sequence as represented by SEQ ID NO: 4 as follows:

Seq. ID NO. 4: 14-3-3 protein beta/alpha

ORIGIN

1 gtggagctac cgccaccgcc gccgccgatt ccggagccgg ggtagtcgcc gccgccgccg

61 ccgctgcagc cactgcaggc accgctgccg ccgcctgagt agtgggctta ggaaggaaga

121 ggtcatctcg ctcggagctt cgctcggaag ggtctttgtt ccctgcagcc ctcccacggc 181 agagtctcca gagatttggg ccgctacaaa aagtgcattt tgcccattcg gctgtggata

241 gagaagcagg aagagcactg gacttggagt cagggaatga caatggataa aagtgagctg

301 gtacagaaag ccaaactcgc tgagcaggct gagcgatatg atgatatggc tgcagccatg

361 aaggcagtca cagaacaggg gcatgaactc tccaacgaag agagaaatct gctctctgtt

421 gcctacaaga atgtggtagg cgcccgccgc tcttcctggc gtgtcatctc cagcattgag 481 cagaaaacag agaggaatga gaagaagcag cagatgggca aagagtaccg tgagaagata

541 gaggcagaac tgcaggacat ctgcaatgat gttctggagc tgttggacaa atatcttatt

601 cccaatgcta cacaaccaga aagtaaggtg ttctacttga aaatgaaagg agattatttt

661 aggtatcttt ctgaagtggc atctggagac aacaaacaaa ccactgtgtc gaactcccag 721 caggcttacc aggaagcatt tgaaattagt aagaaagaaa tgcagcctac acacccaatt

781 cgtcttggtc tggcactaaa tttctcagtc ttttactatg agattctaaa ctctcctgaa

841 aaggcctgta gcctggcaaa aacggcattt gatgaagcaa ttgctgaatt ggatacgctg

901 aatgaagagt cttataaaga cagcactctg atcatgcagt tacttaggga caatctcact

961 ctgtggacat cggaaaacca gggagacgaa ggagacgctg gggagggaga gaactaatgt 1021 ttctcgtgct ttgtgatctg ttcagtgtca ctctgtaccc tcaacatata tcccttgtgc

1081 gataaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa

In one embodiment, the fibroproliferative disorder is associated with an increased deposition of collagen and/or other extracellular matrix proteins. In one embodiment the fibroproliferative disorder is selected from the group comprising fibrosis disorders, in particular fibrosis of liver, kidney, lung, bowel, heart, pancreas, peritoneum, skin, and keloid formation and hypertrophic scarring. Preferably, the fibroproliferative disorder is selected from the group comprising hypertrophic scarring and keloid formation, wherein more preferably, the hypertropic scarring is due to surgery, thermal injury and deep trauma. In a preferred embodiment, the disorder is a thickening of airways/airway fibrosis seen in asthma patients that is associated with accumulation of ECM (namely collagen). Accordingly, in one embodiment there is provided the use of a 14-3-3 protein, derivatives and fragments thereof, and a cDNA in a form of sense and anti-sense in treating asthma patients to reduce airway fibrosis. The extracellular matrix proteins may be selected from the group comprising fibrin, elastin, proteoglycans, keratin, collagens, decorin, fibronectin, and MM PS).

In one embodiment, the mammalian 14-3-3 protein or its functional analogues is recombinantly produced or isolated from tissue and/or body fluids. In another embodiment, the recombinantly produced mammalian 14-3-3 protein is a fusion protein comprising 14-3-3 protein and a fusion peptide. In another embodiment, the recombinantly produced mammalian 14-3-3 protein is 14-3-3 protein devoid of any fusion peptide. In one embodiment, administering occurs prior to the fusion peptide has been cleaved off, or administering occurs without cleaving off the fusion peptide. The fusion peptide may have a sequence comprising RGD.

In one embodiment, administering occurs by topical application of the protein, topical and/or systemic injection of the protein and/or expression of the protein from the nucleic acid encoding for it, wherein preferably, for expression of the protein from the nucleic acid encoding for it, the nucleic acid is brought into a cell by a process selected from the group comprising transfection, transformation, electroporation, and DMA- injection. In one embodiment, the topical injection is a subcutaneous injection, in particular an intradermal injection. In one embodiment, administering occurs by application of specific cells expressing and secreting a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID No: 1 or 2, and/or its functional analogues. The mammal may be a human being.

In accordance with another aspect of the present invention, there is provided a method of increasing collagen content of the skin and/or for the prevention, alleviation and/or treatment of skin having a low collagen content, comprising administering an antibody or binding fragment thereof, wherein the antibody or fragment specifically binds to a 14-3-3 protein, its receptor, or an active fragment thereof, or administering a nucleic acid that is antisense to a nucleic acid encoding for said 14-3-3 protein or administering siRNA directed towards a nucleic acid encoding for a 14-3- 3- protein. Use of an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein to increase the collagen content of a mammal's skin is likewise provided.

Such KDAF neutralizing/blocking compounds are likewise useful in the treatment of collagenase-induced non-healing wounds, collagenase induced nerve degenerative disorders and neuronal damaging in the brain and spinal chord.

In one embodiment, the mammalian 14-3-3 protein is selected from the group comprising 14-3-3 beta/alpha protein, 14-3-3 epsilon protein, 14- 3-3 eta protein, 14-3-3 sigma protein, 14-3-3 tau/theta protein and 14-3-3 zeta/delta protein. Preferably, the mammalian 14-3-3 protein is a human 14-3-3 sigma protein. In one embodiment, the mammalian 14-3-3 protein has a sequence as represented by SEQ ID NO: 1. In another embodiment, the 14-3-3 protein has a sequence as represented by SEQ ID. NO. 2.

In one embodiment, the nucleic acid encoding for said mammalian 14-3-3 protein has a sequence as represented by SEQ ID NO: 3. In another embodiment, the nucleic acid encoding for said 14-3-3 protein has a sequence as represented by SEQ ID. No. 4.

In one embodiment, the skin is ageing and/or wrinkled skin, for example caused by radiation treatment.

In another embodiment, the mammal is a human being. It has also surprisingly been found that cells, which express or which express and secrete a mammalian 14-3-3 protein can be used for screening methods directed at identifying compounds that are capable of increasing the collagen content of the skin. To this end, these cells are incubated with a potentially active candidate compound whereafter the expression and/or secretion of the 14-3-3 protein is measured.

Alternatively a 14-3-3 protein is incubated together with cells expressing collagenase, in combination with a potentially active compound, whereafter the expression of collagenase is measured. The idea behind such a screening method is that an active compound, "active" meaning it will increase the collagen content of the skin, reduces/inhibits the expression, or secretion of the 14-3-3 protein whereby the induction of collagenase expression is reduced. In an alternative embodiment a potentially active compound is incubated together with a 14-3-3 protein and cells expressing collagenase, whereupon the expression of collagenase is measured. In that case if the potentially compound is an active compound it will interfere with the stimulating effect that the 14-3-3 protein has on the expression of collagenase. In both cases the result will be that such a compound has the effect that the collagen content of an association of cells, for example the skin, will be increased.

Therefore, in accordance with another aspect of the present invention, there is provided a method of screening for compounds that are capable of increasing the collagen content of the skin, comprising: - providing cells expressing or expressing and secreting a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID NO: 1 , or SEQ ID. NO. 2, or a functional analogue thereof, or an active fragment thereof,

- incubating said cells with a potentially active compound, - measuring the expression and/or secretion of said protein, or

- providing a mammalian 14-3-3 protein, in particular a mammalian 14-3-3 protein as represented by SEQ ID. NO: 1 , or SEQ ID. NO. 2, or a functional analogue thereof, or an active fragment thereof, - incubating said protein with cells expressing collagenase, together with a potentially active compound,

- measuring the expression of collagenase, an active compound being a compound that reduces/inhibits expression or secretion of said protein, or a compound that reduces/inhibits expression of collagenase.

In another embodiment, the cells expressing or expressing and secreting a mammalian 14-3-3 protein, or a functional analogue thereof, are keratinocytes and epithelial cells. The cells expressing collagenase may be fibroblasts.

Compounds identified by the screening method according to the present invention are likewise provided. In accordance with another aspect of the present invention, there is provided use of a 14-3-3 protein, or a functional analogue thereof including any active fragment thereof, or a nucleic acid encoding for such a 14-3-3 protein, in the manufacture of a medicament for the treatment of fibroproliferative disorders. 14-3-3 proteins and their analogues and nucleic acid sequences can be used for the manufacture of a medicament for the treatment of fibroproliferative disorders, in particular fibrosis of various internal organs, including liver, kidney, lung, bowel, heart, pancreas, fibrosis of vessels and airways, peritoneum, skin, and for the treatment of kiloid formation and hypertrophic scaring. To this end, the 14- 3-3 protein is topically or systemically administered for example by injection into the skin, preferably by subcutaneous injection, even more preferably by intradermal injection. The 14-3-3 protein can be recombinantly produced or it can be isolated from tissue and/or body fluids. If recombinantly expressed, it can be expressed in eukaryotes and/or prokaryotic cells, in particular in yeast and/or E. coli. Furthermore, if recombinantly expressed, it can be expressed as a fusion protein, for example a GST-fusion protein, wherein the fusion peptide has the function of facilitating the purification protocol and, after purification, is cleaved off. Alternatively the fusion peptide may serve the purpose of targeting the 14- 3-3 protein to which it is fused to its particular site of action. For example a fusion peptide, containing the sequence of an active fragment of the 14-3- 3 protein may serve the purpose of facilitating the binding and/or uptake by fibroblasts.

Likewise, an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein, or of a siRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein, or a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein, can be utilized in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.

Particular aspects for the methods and uses are set out in the Examples including the materials and methods as set out below. Examples Example 1

Clinical Specimens and Cell Culture. To establish fibroblast cultures, following informed consent, either fetal foreskin or normal skin punch biopsies, obtained from adult patients undergoing elective reconstructive surgery, were collected individually and washed three times in sterile Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, New York) supplemented with an antibiotic-antimycotic preparation (100μg/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B) (Gibco). Skin punch biopsies were obtained according to a protocol approved by the University of Alberta Hospitals Human Ethics Committee. Cultures of fibroblasts were established as previously described in Reference (1). Upon reaching confluence, the cells were released by trypsinization, split for subculture at a ratio of 1 :β, and reseeded into 75-crn^ flasks. Fibroblasts at passages 3-7 were used in fibroblasts/keratinocyte co- culture system.

To establish cultured keratinocytes, the procedure of Rheinwald aηd Green (Reference 2) was used for cultivation of human foreskin keratinocytes using serum-free keratinocyte medium (GIBCO) supplemented with bovine pituitary extract (50 μg/mL) and EGF (5μg/mL). Primary cultured keratinocytes at passages 3-5 were used. Keratinocytes were then grown either alone or in the upper chamber of a co-culture system with dermal fibroblasts in the lower chamber. In this system any factor released from keratinocytes can diffuse through the 0.4 μm porous membrane separating the two chambers. Example 2

Extraction of Cellular RNA and Northern Analysis. To identify the presence of any factor, which might function as anti-fibrogenic factor for dermal fibroblasts, keratinocytes on the top chamber and fibroblasts from the bottom chamber were harvested separately and lysed in 6 mL of guanidinium thiocyanate (GITC) as previously described in Reference (3). Total RNA was extracted by the GITC/CsCI procedure described before in Reference (4), separated by electrophoresis and blotted onto nitrocellulose filters. Filters were then baked under vacuum for 2 hours at 80°C and pre-hybridized in a solution containing 50% formamide, 0.3 M sodium chloride, 20 mM Tris HCI (pH 8.0), 1 mM EDTA, 1x Denhardt's solution. Hybridization was performed in the same solution at 45°G for 16- 20 hours using cDNA probes for either human collagenase or 18 S. The probes were labelled with 32p__α_< cτP (DuPont Canada, Streets Ville, Mississauga, Ontario, Canada) by nick-translation. Autoradiography was performed by exposing Kodak X-Omat film to the nitrocellulose filters at - 70°C in the presence of an enhancing screen. The cDNA probes for collagenase and 18 S ribosomal RNA were obtained from American Type Culture Collection (Rockville, MD, USA). Example 3

Collagenase Activity. To determine the effects of KDAF on collagenase activity, conditioned medium from either keratinocytes alone, fibroblasts alone or fibroblasts co-cultured with keratinocytes was replaced with serum-free medium supplemented with 25 u/ml conconavalin A (to increase enzyme production) and 0.5%(w/v) lactalbumin hydrolysate (Dibco). After 2 days the conditioned medium from each experimental condition was collected, centrifuged at 1000X g for 10 min, and stored at 4°C. Collagenase assay was carried out as described elsewhere (Reference 5) with slight modifications. Proteins in conditioned medium were precipitated by ammonium sulphate and the precipitates collected by centrifugation, dissolved in assay buffer (0.05M Tris, 0.2M NaCI, 0.005M CaCI₂, 0.02% sodium azide, pH 7.4) and then dialyzed overnight against 4 litters of the same buffer. The final volume of each sample was adjusted according to the cell number. Procollagenase was activated proteolytically with trypsin (10 μg/ml) and soybean trypsin inhibitor (100 μg/ml) was used to inactivate the trypsin. Acetic acid soluble collagen (50 μg in 25 ul) from bovine skin was incubated with the activated enzyme solution in the presence of 1 M glucose for 15-24 hrs. The products from digested collagen were then separated by electrophoresis using 5% acrylamide gel containing sodium dodecyl sulphate (SDS). The gels were stained with Coomassie blue and the β1 , 1 (3/4) and β1 , 2(3/4) fragments were quantified using a computerized scanner (Macintosh Colour One Scanner) and imaging program (NIH Image). Example 4

Identification and Characterization of a Keratinocyte Derived-Anti- Fibrogenic Factor. As the factor of interest present in the KCM possesses a collagenase stimulating activity in fibroblasts, which favours resolution of fibrogenic condition such as hypertrophic scarring, we also refer to this protein as a Keratinocyte Derived Anti-fibrogenic Factor ("KDAF").

To identify this factor, we have established a keratinocyte/fibroblast co-culture system in which keratinocytes and fibroblasts are grown in the upper and lower chambers of this system, respectively. As a permeable membrane separates these cells, fibroblasts in the lower chamber can be exposed to any soluble factor, which may release from keratinocytes. As an index for anti-fibrogenic effects of keratinocyte-derived factor(s) on dermal fibroblasts, total RNA was extracted from fibroblasts grown in the lower chamber and the expression of collagenase mRNA was evaluated by Northern analysis.

Fibroblasts grown alone and fibroblasts grown in a fibroblast/fibroblast co-culture system were also used as a control. Fig. 1 illustrates the expression of collagenase by fibroblast with human dermal keratinocytes. In Lane 1 , RNA from keratinocytes is shown. In Lane 2, RNA from fibroblasts co-cultured with keratinocytes is indicated. In Lane 3, RNA from fibroblasts cultured alone is shown. As is shown in Fig. 1 , collagenase mRNA expression is significantly increased in fibroblasts grown in this system relative to fibroblasts grown either alone or in fibroblast/fibroblast co-culture system. This differential expression is specific as the intensity of 18S ribosomal RNA was relatively similar in all samples (18S band). The effect of KDAF on expression of collagenase mRNA was dose dependent as is shown in Fig. 2, where expression of collagenase mRNA and 18 S Ribosomal RNA(control for RNA loading) by dermal fibroblasts which received various % of keratinocytes conditioned medium was observed.

The collagenase stimulatory effect of keratinocyte conditioned medium (KCM) at different time interval following an addition of test medium (49% serum free medium + 49% DMEM and 2% Fetal bovine serum) was also evaluated. Fig. 3 illustrates the expression of collagenase and 18S ribosomal RNA (control) by dermal fibroblasts received keratinocyte conditioned media from keratinocytes grown in test media at the indicated time intervals. The results shown in Fig. 3 indicate that test medium induced keratinocyte differentiation increases the expression of KDAF at the later time points of differentiation. This activity was also found in both 65 and 95% ammonium sulphate precipitable proteins present in keratinocyte conditioned medium as is illustrated in Fig. 4. Figure 4 illustrates that KDAF present in keratinocyte-conditioned medium can be precipitated by ammonium sulfate. KCM was precipitated by either 65 or 95% ammonium sulfate and protein concentration was determined. Dermal fibroblasts were then treated with KCM (Lane 1 , positive control), non-conditioned medium (NCM) (Lane 2, negative control), either 10 (Lanes 3 and 5) or 100 (Lanes 4 and 6) μg of precipitated proteins for 24 h. Total RNA was then extracted individually and subjected for dot blot analysis. The blots were initially hybridized with collagenase cDNA and subsequently with cDNA specific for 18 S ribosomal RNA used as a control for RNA loading. We noted that either 65% or 95% ammonium sulfate precipitated KDAF stimulates the expression of collagenase mRNA in a dose dependent fashion. To examine whether KDAF is also functional for fibroblasts embedded within a collagen gel (3D condition), a skin substitute was made. Keratinocyte layers and fibroblasts populated in collagen +GAG gel were used an epidermal and dermal components of this skin substitute, respectively (Fig. 5). Fig. 5 shows a dermal-epidermal skin substitute prepared from keratinocyte/fibroblasts collagen-GAG gel. Panel A shows a section of dermal-epidermal skin substitute stained with H&E . Panel B shows expression of collagenase and 18 S ribosomal RNA from fibroblasts in collagen matrix (lane 1 ), keratinocytes in epidermal layer (lane B) and fibroblasts alone on plastic (lane 3). This finding shows that KDAF is functional on fibroblasts grown in a three dimensional environment. The collagenase expression in fibroblasts populated collagen gel in the presence and absence of epidermis was then evaluated. As shown in Fig 5, the expression of collagenase mRNA significantly increased only in those fibroblasts that were exposed to keratinocytes as an epidermal layer.

In Fig. 6, the efficacy of KADF of keratinocyte conditioned medium on collagenase activity was also evaluated and the results show that KADF significantly breaks down type I collagen to its 1/4 and 3/4 fragments of the α (α1.1 and α1.2) and β (β1.1 and β1.2) chains of type I collagen which was used as an index for collagenase activity. Collagenase activity in conditioned medium obtained either from keratinocytes alone (K), fibroblasts alone (F) or keratinocyte/fibroblast (K/F) co-culture was evaluated in triplicate using type I collagen as a substrate. Type I collagen with no treatment (collagen) was also included as a control. Only conditioned medium derived from keratinocytes was able to markedly break down type I collagen to its 1/4 and 3/4 fragments of the α (αl.1 and αl .2) and β (β1.1 and β1.2) chains of type I collagen. Example 5

Estimation of the Apparent Molecular Weight of KDAF. To provide a rough estimate of the molecular weight of KDAF, KCM was passed through 50 and 30 kDa cut-off filters of the Centricon Tubes (Amicon) and portions of unfiltered material, filtrate and retentate were individually analyzed. When 50 kDa cut-off filters were used, collagenase stimulating activity was found in the unfiltered KCM, retentate and filtrate treated fibroblasts relative to the corresponding samples obtained from non-conditioned medium (Data not shown). Collagenase stimulatory effect of KDAF was not due to RNA loading as the pattern of 18S ribosomal RNA was relatively the same in all samples (data not shown). These findings collectively suggest that both retentate and filtrate of 50 kDa cut-off KCM possess collagenase stimulatory effects in dermal fibroblasts. To further narrow down the size of this factor, a 30 kDa retentate and filtrate of KCM was tested on collagenase expression in dermal fibroblasts. The results clearly show the presence of collagenase stimulating activity in both unfiltered KCM and retentate, but not in filtrate-treated dermal fibroblasts. This is a clear indication that the apparent size of this factor is ranged between 30 and 50 kDa. The shelf life of the protein was seen to be very good as high temperature up to 50° C for 30 min, room temperature up to 48 hr, and 5 times freeze- thawing did not abrogate the activity of this protein. Example 6

Fractionation of Proteins Obtained from ECeratinoc e-Conditioned Media. The media collected from keratinocyte-conditioned medium every 48 h over a 24-day period was subjected to a 65% ammonium sulphate precipitation followed by centrifugation at 10, 000 g for 15 min. The pellet was re-suspended in a minimum volume of buffer A (10 mM sodium phosphate, pH 7.3, 150 mM NaCI, 4 mM 2-mercaptoethanol, protease inhibitor cocktail (Sigma)), and dialyzed overnight at 4°C in the same buffer. Approximately 200 μg of protein in 50 ul was loaded onto a Superdex-75 PC 3.2/30 gel filtration column attached to a SMART micro- purification system (Amersham Pharmacia BioTech). Protein was eluted with buffer A and twenty-five 80-ul fractions were collected. The fractions were examined by electrophoresis on a 10% SDS-polyacrylamide gel and staining with Comassie Blue. Example 7

Mass Spectrometry . a)Proteins in candidate bands excised from SDS/polyacrylamide gels were subjected to trypsin digestion according to a published procedure (Shevchenko et al, Anal. Chem, 68: 850-858, 1996) and MS analysis (Dai et al, Anal Chem. 71 :1087-1091, 1998). Matrix assisted laser adsorption mass spectrometry (MALDI MS) and matrix assisted laser adsorption/ionization post source decay mass spectrometry (MALDI PSD MS) were performed on a Voyager Elite MALDI MS instrument (Voyager Elite, PerSeptive Biosystem, Inc., Framingham, MA) equipped with a delayed extraction (DE) device. A two-layer method was used for MALDI MS analysis in which 1 to 2 ul of first layer solution [10 mg/ml of 4-hydroxy-alpha-cyanocinnamic acid (HCCA) per mL of 20% methanol/acetone (v/v)] was deposited onto a probe tip, and evaporated to form a thin matrix layer, and then 0.5 - 1 uL of gel extract from 50% acetonitrile or 40% methanol saturated by HCCA was deposited onto the first layer, allowed to air dry, and washed three times with water. The PSD spectra were recorded in the PSD mode of the Voyager Elite instrument. Nanoelectrospray (NanoES) ion trap MS was performed on an Esquire-LC ion trap spectrometer (Hewlett-Packard, Reno, NV) with NanoES interface. Spectra were acquired over the mass range 200 to 2200 Da. b) Samples from candidate protein bands were prepared according to ACB (Alberta Cancer Board) Proteomics Resource Laboratory's protocols (Karimi-Busheri et al, J. Cell. Biochem, 64:258-272, 1997]. In brief, these protocols include washing, reduction, alkylation, tryptic digestion and extraction of tryptic peptides from the gel spots. Peptide extracts were analyzed on a Bruker REFLEX III (Bremen/Leipzig, Germany, Serial#: FM 2413) time of flight mass spectrometer using MALDI in positive ion mode. Obtained peptide maps were used for database searching to identify proteins. Furthermore, for each sample 1-3 selected peptides were fragmented using MALDI MS/MS analysis done on a PE Sciex API-QSTAR pulsar (MDS-Sciex, Toronto, Ontario, Canada, Serial# K0940105). The obtained partial sequence information for each peptide was used to either confirm or correct the previously obtained results from the peptide map search.

For each 1 D gel band at least one protein was identified. The results identified the candidate protein band as being 14-3-3 sigma protein, also known as stratifin. Example 8

Cloning, Expression and Purification of KDAF. For KDAF sigma (14-3- 3 protein sigma isoforrn) cloning, total RNA was prepared by the acid- guanidium-phenol-chloroform method from either human keratinocytes (for KDAF sigma) or fibroblasts (for 14-3-3 protein α/β which we now refer as KDAFα/β). Each cDNA was then synthesized with oligo (dt) primer and MMLV reverse transcriptase (Gibco-BRL)). Samples were then incubated at 42°C for 60 minutes, and the reaction was terminated by heating at 70°C for 15 minutes and followed by rapid chilling on ice. PCR reaction was carried out with either KDAF-σ primer sense: 5'-

GAATTCCCCAGAGCCATGGAGAGAGCC-3' (SEQ ID. NO: 5); antisense: 5'-CTCGAGTGGCGGGCAACACTCAGCTC-3' (SEQ ID. NO: 6), or KDAFα/β primer sense: 5^!-GGATCCACAATGGATAAAAGTGAG-3' (SEQ ID. NO: 7); antisense: 5'-CTCGAGAGCACGAGAAACATTAGT-3' (SEQ ID. NO: 8). PCR reaction was carried out for 30 cycles and the PCR products were separated by electrophoresis on 1 % agarose gel. The separated DNA products were stained with ethidium bromide and DNA bands were visualized under UV light.

DNA in agarose gel was purified with QIAEX II gel extraction kit according to manufacturer's instructions (Qiagen). Purified DNA was then digested with either ECoRI/Xhol (for KDAF-σ) or BamHI/Xhol (for KDAFα/β) for 2 hours at 37°C. The digested products were separated by electrophoresis on 1 % agarose gel and specific DNA band related to either KDAF-σ or KDAF-α/β was purified by QIAEX II gel extraction kit. Finally, the purified DNA was ligated into a pGEX-6P-1 expressing vector using GST protein (Amersham/Pharmacia Biotech). For Bacteria transformation, the ligated products were transformed to competent XL0blue-1 cells with regular heat shock transformation method. Positive clones were identified by the size of restriction enzyme digested products. DNA sequence was confirmed by fluorescence dNTP sequence analysis. The plasmid DNA containing either KDAF-σ or KDAF- α/β was transformed into protein expressing bacteria BL-21 (DE3) (Novagene).

For protein expression, single positive clone was grown in 100 ml of LB medium containing 50 μg/ml of ampicillin for 4-6 hours at 29°C until an ODβoonm of 0.4-0.6 was reached. Bacteria were then diluted to 1 :10 with fresh LB medium grown in the presence of 0.1 mM of IPTG for 24 hrs.

For protein purification, Bacteria were collected by centrifuging and lysed with 50 mM Tris-HCI (pH7.4) containing 10 mM EDTA, 5mM EGTA, Protease cocktail (Sigma), 1% Triton X-100, and 0.5% IGEPAL CA630. Cell lysate was passed through Glutathione Sepharose 4B affinity column. The column was then washed with PBS containing 0.1 %Triton X-100 until the OD 2₈₀nm equal to 0 was reached. Either free KDAF-σ or free KDAF-α/β was eluted by PreScission protease digestion according to the manufacturer's instruction (Amersham/Pharmacia Biotech.). Purified proteins were dialysized against PBS and concentrated by Centricon

(Millipore) and used to treat different cell strains. The results of Figs. 7 A and B clearly show the high purity of the recombinant of both sigma and α/β KDAF proteins. In Figure 7A, lanes contain the following: Lane M shows pre-stained SDS-PAGE standards (low range); Lane 1 shows GST-sigma KDAF expressed in BL-21 -DE3 cells; Lane 2 shows extraction of GST-KDAF expressed in BL-21 -DE3 cells; Lane 3 shows purified GST-KDAF fusion protein by Glutathione affinity purification; Lane 4 shows purified KDAF.

In Figure 7B, lanes contain the following: Lane M shows pre- stained SDS-PAGE standards (low range); Lane 1 shows GST-KDAF expressed in BL-21 -DE3 cells; Lane 2 shows extraction of GST-KDAF expressed in BL-21 -DE3 cells; Lane 3 shows purified GST-α/β KDAF fusion protein by Glutathione affinity purification; Lane 4 shows purified α/β KDAF Example 9

Validation of the Collagenase Stimulatory Effects of Recombinant KDAFs. To demonstrate whether the purified KDAFs function as collagenase stimulating factors for dermal fibroblasts, a characteristic feature of keratinocyte conditioned medium, Northern analysis of total RNA extracted from cells treated with various concentration of KDAF- sigma for 24 hrs was carried. The results shown in Fig. 8 demonstrate more than 20 fold increase in collagenase mRNA expression in KDAF treated cells relative to untreated control. In Fig. 8, fibroblasts were treated with keratinocyte conditioned medium (KCM), DMEM +2%FBS (C), or 0.01 , 0.1 , 0.5, 1.0, 2.5, 5.0 (lanes 1-6) μg of purified recombinant KDAF for 24 hrs. Total RNA was extracted and collagenase mRNA was detected by Northern analysis. KDAF increased the expression of collagenase mRNA more than 10 fold relative to that of control.

This increase was even greater than that shown for keratinocyte conditioned medium treated cells (Fig. 2, dose response experiment).

Further, the results of a recovery experiment demonstrated a KDAF lasting effect for only 24 hr upon, KDAF removal from conditioned medium (Fig. 9). Fibroblasts were treated with either vehicle (C), or 2.4 μg/ml KDAF for 24 hr and then the KDAF was removed and cells were harvested at indicated time intervals. Total RNA was then extracted and evaluated for collagenase and type l collagen mRNA. It was observed that the level of collagenase but not collagen gradually return to normal value after KDAF removal, i.e., that the collagenase stimulatory effect of KDAF was not permanent.

In Fig. 10, time dependent keratinocyte conditioned medium induced the expression of collagenase mRNA. Dermal fibroblasts were treated with conditioned medium collected from keratinocyte grown in 50/50 medium and cells were harvested at indicated time intervals and collagenase mRNA was evaluated by Northern analysis. Pattern of 18 S shows that this increase is not due to RNA loading. KCM increased the collagenase mRNA expression in fibroblast as early as 12 hr post treatment and its level reached plateau within 24 hrs.

In Fig. 11 , using the same validation assay, it was also found the KDAF α/β increases the expression of collagenase mRNA. A dose dependent α/β form of KDAF induced the expression of collagenase (A) but not type I collagen (B) mRNA. Fibroblasts were treated with indicated concentrations of α/β form of KDAF for 24 hr. Cells were then harvested and total RNA was evaluated for collagenase and type I collagen expression by Northern analysis. For comparison, the effects of KDAF on collagenase mRNA expression (C ) is also shown, α/β form of KDAF increased the expression of collagenase mRNA, (Panel A), but not collagen, in a dose dependent fashion; this effect was reduced in that instance relative to KDAF (Panel C)

As reviewed by Yaffe MB, (FEBS Letter 513, 53-57, 2002), there is a very high homology (more than 70%) amongst the members of protein 14-3-3 proteins, It is reasonable to predict that other KDAF isoforms would likewise function as collagenase stimulating factors for dermal fibroblasts and possibly other cell types as well. Example 10 Preparation of Active fragments of 14-3-3 protein. Purified KDAF (protein 14-3-3 sigma) was first treated with iodoacetamide and then digested by trypsin. The digests were fractionated using reverse phase HPLC (2.1 mm d 8 column, flow rate 0.2 mL/min). In order to eliminate the interface of triton x-100. The samples were then cleaned by hpl ziptip. The fractions were collected every minute and dried in the speed vac. with a liquid nitrogen trap installed. The fractions were individually added to cultured fibroblasts and total RNA was extracted after 24 hrs. The expression of collagenase mRNA was then evaluated as an index for KDAF activity. From 70 fractions collected as above, fraction numbers 12, 40 and 50 were identified to be active.

Peptides contained in any of the fractions can be synthetically produced using conventional techniques well known to those skilled in the art. In one case, a peptide comprising 8 amino acids was isolated from a fraction and was found to be active upon testing with the methods described herein. Example 11 Preparation of antibodies. Rabbit anti-human polyclonal antibody that specifically reacts with protein 14-3-3 sigma were raised using conventional methods well known to those skilled in the art. As in example 10, when the active fractions of protein 14-3-3 were identified and sequenced, either poly or monoclonal anti-bodies will raised and used as an anti-KDAF factor in both in vitro and in vivo.

Three mg of the purified KDAF (14-3-3 sigma protein) was dissolved in 1 mL of 50 mM Tris-HCI (pH 7.5), 150 mM NaCI, 1 mM EDTA, I mM DTT and 0.01%TritonX-100 buffer. After 6 (first bleeding) and 8 (second bleeding) weeks, rabbit anti- human KDAF antibody was obtained (Washington Biotechnology, Inc, (6200 Freeport Center, Baltimore, MD, 21224.6506, USA)). The rabbit IgG was purified by HiTrap Protein A affinity column (Amersham/Pharmacia Biotech). IgG was then eluted with 0.1 M sodium citrate (pH. 3.5) according to manufacture's instruction. Using this procedure, we were able to produce 25 mg of IgG from 25 mL of rabbit serum. The specificity of this antibody was confirmed by western blot analysis. Example 12

Gene Silencing using siRNA. A standard protocol established by the Gene Therapy System Inc. (GTS) and a commercially available Dicer siRNA Construction Kit is used to mimic the natural human dicer enzyme to cleave in vitro transcribed dsRNA template (protein 14-3-3 mRNA ) into a pool of functional 22 bp siRNA. Example 13 Determination of collagenase stimulatory active site of KDAF. Example 10 was repeated and more data was gathered. As shown in Figure 12, from a total of 60 new fractions examined, fractions numbers 12, 41 , 44, 47and 50 of the new fractions showed strong collagenase stimulatory effects in fibroblasts. However, it is not known whether this is due to the presence of several active sites within the KDAF molecule or this is simply due to sequence homology amongst the peptides present in these fractions. The lower panel shows the pattern of 18 and 28 S ribosomal RNA for the same blots and is used as a total RNA loading control in Northern analysis.

The findings of this study shows that the intact KDAF molecule is not required for its full collagenase stimulating activity in fibroblasts.

Example 14

Monomeric form of KDAF possesses collagenase stimulatory effects in dermal fibroblasts. To determine whether dimeric or monomeric forms of KDAF possess collagenase stimulatory effects in fibroblasts, a serial deletion of KDAF cDNA sequence was performed so that the KDAF protein became 15 (d15), 26 (d26), 50 (d50), 75 (d75), and 100 (d100) amino acids shorter at their N-terminal. The corresponding cDNA for each of these peptides was then cloned and expressed in E.coli. As shown in Fig. 13 A, the purified d15, 26, 50, 75 and d100 amino acid deleted KDAF proteins were then analysed on native gel (Upper panel) and SDS-PAGE (lower panel) for confirmation. As a protein marker, the protein standard (lane 1 ) was also included, trypsin inhibitor with apparent MW of 21 kDa (lane 2) and bovine serum albumin (BSA) with apparent MW of 66 kDa (lane 3) in our samples run on a native gel. The purified proteins corresponding to each of these deleted constructs were then individually added to fibroblasts and the levels of MMP-1 protein were evaluated. Results shown in Fig. 13 B, clearly indicate that deletion of up to 75 amino acids is not critical to the KDAF collagenase stimulatory effect. However, when 100 amino acids of N-terminal KDAF protein was deleted, its collagenase stimulatory effect in fibroblasts was sharply reduced. It is well known that amino acids 1-16 at the N-terminal of the two monomers of 14- 3-3 sigma (KDAF-sigma) are needed for dimerization of this protein. As d50 and d75 KDAF proteins which lack the N-terminal sequences for dimerization of the two monomers still possess collagenase activity, dimerization may not be critical for the collagenase activity of KDAF in dermal fibroblasts. In fact, this finding is consistent with those shown in Figure 14 revealing that some fragments of KDAF generated by trypsin digestion still possess collagenase stimulatory effects for fibroblasts. Thus the findings of Figures 12 and 13 clearly show that not only a monomeric form of KDAF, but also any fragment of KDAF containing the active site of KDAF with the ability to interact to cell surface receptors are sufficient to stimulate the expression of collagenase in dermal fibroblasts.

Example 15

KDAF is Present in Sera Collected from Rat and Bovine and Functions as a Circulating Collagenase Stimulating Factor. To examine whether KDAF is present as a circulating collagenase stimulating factor in serum, sera from rat, bovine and human were collected and evaluated for the presence of KDAF protein by western analysis. We also included rat pituitary extract as a possible source of circulating KDAF. To achieve this, human dermal fibroblasts were treated with various concentrations (0, 0.05, 0.1 , 0.5, 1.0, 5.0 %) of FBS and bovine pituitary extract (BPE) in DMEM for 24 hr. Total RNA was extracted and evaluated by Northern analysis using either cDNA probe for collagenase (top panel) or 18 S ribosomal RNA (bottom panel). Please note that the levels of collagenase mRNA expression remarkably increases in response to either serum or pituitary extract relative to that of untreated control (Fig. 14A). This finding clearly indicates that serum and pituitary extract possess collagenase stimulatory effects in dermal fibroblasts. To confirm whether this collagenase stimulatory effects is due to the presence of KDAF in serum and pituitary extract, the sera and pituitary extracts from rat and bovine were collected and 50μg of their corresponding total protein /sample was subjected to SDS-PAGE. As shown in Fig. 14B, the amount of KDAF protein was visualized by Western blotting using our recently raised anti-KDAF antibody. Two samples of our recently purified recombinant KDAF were also loaded as positive controls. To evaluate the biological role of serum KDAF in expression of collagenase in dermal fibroblasts, FBS proteins (3 μg/lane) were run on SDS-PAGE and fractionated proteins were identified according to their MW in reference to MW marker. As the size of KDAF is determined to be 28-30 kDa, protein fractions containing KDAF with apparent MW of 28-32 kDa (K+) and those without KDAF with MW of 45-55 kDa (K-) were excised and recovered by electroelution. As shown in Fig. 15, proteins of these fractions were either run in a SDS-PAGE using un-fractionated FBS sample as a control (panel A) or used to treat dermal fibroblast to evaluate its collagenase activity (Panel B). Proteins in panel A were then blotted and evaluated for the presence of KDAF by Western blot analysis. Note, KDAF antibody identified a band with apparent MW of 30 kDa only in whole FBS sample and K+ fraction (Panel A). Panel B shows the pattern of collagenase mRNA expression and 18 S ribosomal RNA (loading control) in fibroblasts treated with either nothing (C), recombinant KDAF (K), electroelusion buffer (B), various concentrations of proteins in K- fraction (K-) or K+ fraction (K+). Please note that collagenase mRNA expression is markedly increased only in response to either rKDAF or various concentrations of proteins in K+ fraction. Panel C demonstrates the pattern of western blot analysis showing the presence and quantities of KDAF in sera collected from normal, sham operated or hypophysectomized rats. Note that KDAF level is remarkably increased upon removal of pituitary gland (Hypo) relative to that of controls. This indicates that circulating KDAF is regulated by hormones but not released from pituitary gland.

Example 16

Human serum contains relatively high levels of KDAF. In order to examine whether human serum also contains KDAF, five ml of blood was drawn from each of 8 volunteers in our lab with different sex, age and race. As shown in Fig. 16, corresponding sera were separated and 50 μg of total protein /sample was subjected to SDS-PAGE followed by Western blotting (Upper panel) . Purified KDAF was also used as a positive control and Ponceu S staining of the membrane was shown as a loading control (Lower panel). The finding of this experiment clearly shows that human serum contains a relatively high level of KDAF protein. As for rat and bovine sera, KDAF in human serum may function as a circulating collagenase stimulating factor.

Example 17

In vivo administration of KDAF improves healing quality in rat model. To evaluate the healing quality of treated and untreated wounds in rat model, we have compared the histology of KDAF treated and untreated healing wound tissue sections in rat model. A series of circular wounds with 6 mm in diameter were generated on the backs of each of 4 rats. After 5 days, when enough granulation tissue was formed, wounds received either dermal cream (control, or cream) containing 10 μg of our purified recombinant KDAF/gram of cream. Treatment continued up to day 21 post wounding. The same wound sites were taken by the same size punches. Tissue samples were then fixed, sectioned at 6 μm thickness and stained with H&E. As shown in Fig. 17, tissue cellularity and ECM deposition are markedly improved in wounds treated with KDAF relative to those of untreated control. Example 18

A M^'mtur® ©f t^D F and Aspirin (aeetj/Isalicylic; acid) possesses both a collagenase stimulatory and collagen inhibitory effects in dermal fibroblasts. In general, an effective anti-fibrogenic factor for treatment of fibroproliferative disorders such as hypertrophic scarring should possess either a collagenase stimulatory or a collagen (the main ECM component in skin) inhibitory effect for fibroblasts. Thus, any factor with a combination of both should be even more effective than either of these factors used individually. In Figures 1-6 and 8, 9 and 11, it has been demonstrated that both keratinocyte conditioned medium (KCM) and purified KDAF significantly increase the expression of collagenase in dermal fibroblasts. Here, we have supporting evidence that various concentrations of aspirin (acetylsalicylic acid) markedly suppress the expression of collagen type I in dermal fibroblasts. As such a combination of aspirin at final concentration 2.5 mg/ml and KDAF at 2.5 μg/ml not only suppresses the expression of collagen (Fig. 18, Panel A), but also significantly increases the expression of collagenase in dermal fibroblasts. When the same blot was hybridized with a cDNA specific for tissue inhibitor for metalloproteinase -1 (TIMP-1) a slight increase was found in response to aspirin treatment (Fig. 18, Panel B). This finding as well as the loading of 18 and 28 ribosomal RNA shown in Fig. 18, Panel C, indicate that a remarkable suppression of type I collagen mRNA in aspirin treated cells is specific and is not caused by any variation in loading of total RNA. Thus, data shown in Fig. 1-6 as well as those of Fig. 18 clearly indicate that a mixture of these components would be even more effective as antifibrogenic factor for treatment of fibroproliferative disorder in skin. As aspirin has also a keratinolytic effect on skin, topical application of this mixture would diffuse better through the epidermal layer to the dermal portion of skin.

References

All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

1. Ghahary A, Tredget EE, Chang LJ, Scott PG, Shen, Q. Genetically modified dermal keratinocytes express high levels of transforming growth factor-β1 mRNA and protein. J. Invest. Dermatol. 110:800-805, 1998.

2. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-34, 1975.

3. Bauer S. B. Tredget EE, Scott PG, Marcoux Y, Ghahary A. Latent and Active Transforming Growth Factor β1 Released from Genetically Modified Keratinocytes Modulates Extracellular Matrix Expression by Dermal Fibroblasts in a Coculture System J. Investigation Dermatol. 119:456, 2002 4. Ghahary A, Tredget EE, Ghahary AT, Bahar MA, Telasky C. Cell proliferating effects of latent transforming growth factor β1 is cell membrane dependent. Wound Rep. Reg.10:328-335, 2002.

5. Khorramizadeh MR, Tredget EE, Telasky C, Shen Q, Ghahary A. Aging differentially modulates the expression of collagen and collagenase in dermal fibroblasts. Mol. Cell. Biochem. 194:99-108, 1999.

Claims

WHAT IS CLAIMED IS:

1. Use of a 14-3-3 protein or a functional analogue thereof in the treatment of a fibroproliferative disorder in a mammal.

2. The use of claim 1 , wherein the 14-3-3 protein comprises a mammalian 14-3-3 protein.

3. The use of claim 1 , wherein the 14-3-3 protein comprises one of a plant, yeast and fungi 14-3-3 protein.

4. Use of a nucleic acid encoding for a 14-3-3 protein or a functional analogue thereof in the treatment of a fibroproliferative disorder in a mammal.

5. The use of any one of claims 1 to 4, wherein the fibroproliferative disorder is selected from the group consisting of fibrosis disorders, fibrosis of the liver, fibrosis of the kidney, fibrosis of the lung, fibrosis of the bowel, fibrosis of the heart, fibrosis of the pancreas, fibrosis of vessels and airways, fibrosis of the peritoneum, fibrosis of the skin, keloid formation, and hypertrophic scarring.

6. The use of any one of claims 1 to 4, wherein the fibroproliferative disorder is associated with an increased deposition of at least one of collagen and one or more additional extracellular matrix proteins.

7. Use of an antibody or a binding fragment thereof which can specifically bind to a mammalian 14-3-3 protein or immunogenic fragment thereof to increase the collagen content of a mammal's skin.

8. Use of a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein to increase the collagen content in a mammal's skin.

9. Use of a SiRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein to increase the collagen content in a mammal's skin.

10. Use of a 14-3-3 protein in the manufacture of a medicament for the treatment of a fibroproliferative disorder.

11. Use of a functional analogue of a 14-3-3 protein in the manufacture of a medicament for the treatment of a fibroproliferative disorder.

12. Use of a nucleic acid encoding for a 14-3-3 protein or a functional analogue thereof in the manufacture of a medicament for the treatment of a fibroproliferative disorder.

13. Use of an antibody or binding fragment thereof wherein the antibody or fragment is capable of specifically binding to a mammalian 14-3- 3 protein, its receptors or immunogenic fragment thereof, in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.

14. Use of a SiRNA directed towards a nucleic acid encoding for a mammalian 14-3-3 protein in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.

15. Use of a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein in the manufacture of a medicament for the treatment of a condition associated with a decreased level of collagen.

16- A method for decreasing the collagen in a tissue or organ comprising administering an effective amount of a 14-3-3 protein or a functional analogue thereof, or a nucleic acid encoding for said protein.

17. Use of at least one of a 14-3-3 protein, an active fragment thereof, an antibody or binding fragment thereof which is capable of specifically binding to a 14-3-3 protein, a nucleic acid that is antisense to a nucleic acid encoding for a mammalian 14-3-3 protein, and a siRNA directed towards a nucleic acid encoding for a 14-3-3 protein, in the manufacture of a medicament for the treatment of a condition associated with aging improvement in an organ.

18. The use of claim 17, wherein the organ comprises skin.

19. The use of a mixture of a pharmaceutically acceptable weak acid or a derivative thereof and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein in the manufacture of a medicament for the treatment of fibroproliferative disorder.

20. The use of a mixture of acetylsalicylic acid and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein in the manufacture of a medicament for the treatment of fibroproliferative disorder.

21. The use as claimed in claim 19 or 20 wherein the fibroproliferative disorder is hypertrophic scarring.

22. A pharmaceutical preparation for the treatment of fibroproliferative disorders comprising a mixture of a pharmaceutically acceptable weak acid or a derivative thereof and a 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein together with a pharmaceutically acceptable carrier therefor.

23. A pharmaceutical preparation for the treatment of fibroproliferative disorders comprising a mixture of acetylsalicylic acid and 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein ranges together with a pharmaceutically acceptable carrier therefor.

24. A pharmaceutical preparation as claimed in claim 22 or 23 wherein the ratio of weak acid or a derivative thereof to the 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein ranges from 1 :1 to about 1 :1000 on a dry weight basis.

25. A pharmaceutical preparation as claimed in claim 23 wherein the ratio of acetylsalicylic acid to the 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein is 1 :1 on a dry weight basis.

26. A pharmaceutical preparation as claimed in claim 25 wherein the amount of acetylsalicylic acid is 2.5 mg/ml and the amount of 14-3-3 protein, any active fragment thereof, or a functional analogue thereof, or a nucleic acid encoding for a 14-3-3 protein is 2.5 μg/ml.