US20060217310A1

US20060217310A1 - Fibroblast growth factor 1 (FGF-1) used for skin care

Info

Publication number: US20060217310A1
Application number: US11/387,051
Authority: US
Inventors: Ing-Ming Chiu; Toko Wen
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-08-17
Filing date: 2006-03-23
Publication date: 2006-09-28
Also published as: EP1634877B1; US20090048330A1; US20060089402A1; ATE513823T1; EP1634877A1; US7732482B2; US20060052337A1; US7763723B2

Abstract

Provided are compositions comprising a fibroblast growth factor 1 (FGF-1) for skin care, and methods of use of such compositions. The FGF-1 is constructed into a recombinant expression vector, and characterized by the molecular characteristics. The FGF-1 can modulate mitogenic activities and mediate both the FGF-2 and KGF signaling pathway that can promote proliferation of fibroblast cells and epidermal cells. A mutant FGF-1 has the longer half life than wild type has in virtue of its capabilities of anti-oxidization or no further proteolytic degradation.

Description

PRIORITY STATEMENT

This application claims priority under 35 U.S.C. §119 of Taiwanese Patent Application No. TW 094109024, filed on Mar. 23, 2005, in the Taiwan Patent Office, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to a composition comprising a fibroblast growth factor 1 (FGF-1) for producing skin care products.

BACKGROUND

It is well known that as individuals age the rate of epidermal cell replication and desquamation, i.e. turnover of cells, decreases or the epidermis becomes senescent, this process frequently produces a dull, aged appearance. In addition, the vascularity of skin decreases with time and the underlying collagenous framework undergoes structural fragmentation secondary to aging and photo-damage, hence elastosis; as a result, wrinkles and sagging occur.
“Senescence” at the cellular level results from inadequate DNA repair leading to disordered and/or nonexistent cell replication. Loss of mitotic control factors in the nucleus and cytoplasm including disordered nuclear cytoplasmic exchange and permanent closing of microcirculatory capillary beds results in focal cell dropout and loss of cell and organelle membrane function.
The lifetime effects of the damage include wrinkling and hardening of the skin with age. The skin is made up of supportive material, including elastin and collagen. Collagen is a major protein component of the white fibers of connective tissue, such as cartilage and bone. White elastin is the major protein in the connective tissue of large blood vessels in the skin which enables these tissues to stretch and resume their original conformation. Both collagen and elastin contain fibers that are linked together with imide bonds. It is believed that mammalian or human aging involves the oxidation of imide bonds to amide bonds with decreased elastic and flexible properties. A free radical mechanism is involved in wrinkling of the skin and results from the negative effects oxidation products which cause tissue aging.
The fibroblast growth factor family has emerged as a large family of growth factors involved in soft-tissue growth and regeneration. It presently includes several members that share a varying degree of homology at the protein level, and that, with a few exceptions, appear to have a similar broad mitogenic spectrum, i.e., they promote the proliferation of a variety of cells of mesodermal and neuroectodermal origin and/or promote angiogenesis.
The pattern of expression of the different members of the family is very different, ranging from extremely restricted expressions at some stages of development, to rather ubiquitous expression in a variety of tissues and organs. All the members appear to bind heparin and heparan sulfate proteoglycans and glycosaminoglycans and are strongly concentrated in the extracellular matrix.
U.S. Pat. No. 4,695,590 describes a method for retarding aging by administering synthetic chemicals, such as certain hydroxy diphenyl alkyl derivatives, preferably by oral administration. It would be desirable to avoid the internal administration of synthetic chemicals both for convenience and to avoid possible side effects of internally administered synthetic chemicals.
A variety of protein factors are known to be essential to the growth and differentiation of cells including epidermal cells. Many of these proteins extracted from tissues have been identified: such as epidermal growth factor (EGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and the like. U.S. Pat. No. 4,959,353 describes the use of epidermal growth factor for treating corneal wounds and U.S. Pat. No. 5,130,298 describes compositions of epidermal growth factor stabilized against degradation with metal cations and used for treating wounds. U.S. Pat. No. 5,104,977 discloses use of TGF-beta with either TGF-alpha for treating damaged tissue. However, as these patents illustrate, protein growth factors have not been previously shown to decrease epidermal cell senescence in unabraded or nonwounded skin. It had been previously thought that large proteins such as growth factors could not penetrate uninjured or intact skin in order to reach the appropriate basal cell layers to increase cellular replication and thereby decrease skin cell senescence.
It would be desirable to have a simple composition to achieve the skin care effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SDS-PAGE analysis and immunoblotting of recombinant FGF-1. (A) Increasing amount (100, 250, and 500 ng) of newt, bovine and human FGF1, premixed with the same amount of bovine serum albumin, were electrophoresed on a 15% SDS polyacrylamide gel and then silver stained. (B) A duplicate gel of (A) was transferred to a nitrocellulose membrane and immunoreacted with a polyclonal antibody (1:1000, Promega) directed against a peptide derived from bovine FGF1. Target proteins were detected with horseradish peroxidase-conjugated secondary antibodies. The arrow indicates the slower migrating proteins of newt and human FGF-1 and the arrowhead indicates bovine FGF1. Molecular weights were indicated on the left.
FIG. 2 shows mitogenic response of Swiss/3T3 fibroblast cells to bovine and human FGF-1. Swiss/3T3 cells were grown to ˜80% confluency in 24-well plates and then serum starved for 24 h. Bovine (closed circle) or human (open circle) recombinant FGF-1 at the indicated concentration was added directly to the starvation media, and the cells were incubated for 20 h after which time they were pulsed with 1 μCi of [3H] thymidine for 6 h and the amount of radioactivity was determined. Values are the mean cpms of duplicate samples (±SE).
FIG. 3 shows that FGF-1 mediates repression of skeletal muscle differentiation in MM14 cells expression newt and human KGFR while FGF-2 is not responsive to KGFR. The transfected cells were cultured either in the absence of any exogenously added growth factor (filled bars) or in the presence of FGF-1 (open bars), FGF-2 (stippled bars), or FGF-7 (hatched bars). The value obtained for each set of transfected cells cultured in the absence of growth factors was set at 1.0.
FIG. 4 shows CuCl₂treatment of human and bovine wild-type and mutant FGF-1 proteins. Human wild-type ( lanes 1, 2, and 9), human C131S mutant ( lanes 3, 4, and 10), bovine wild type ( lanes 5, 6, and 11), and bovine S131C mutant ( lanes 7, 8, and 12) were incubated in the absence ( lanes 1, 3, 5, and 7) or presence ( lanes 2, 4, 6, and 8-12) of CuCl₂. The reactions were either without (lanes 1-8) or with (lanes 9-12) the addition of dithiothreitol (DTT). The reaction products were separated by 15% nonreducing SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, reacted with an anti-FGF-1 antibody, and visualized with ECL. Molecular mass markers are indicated in kDa.

SUMMARY OF THE INVENTION

This invention provides a composition for promoting proliferation of fibroblast cells or epidermal cells comprising an effective amount of a fibroblast growth factor 1 (FGF-1; SEQ ID NO: 1). This invention also provides a truncated form of FGF-1 representing the natural proteolytic product in human cells, wherein the amino acids No. 2 to No. 15 are deleted (SEQ ID NO: 2). This invention further provides hFGF-1 mutants representing highly anti-oxidization properties comprising the amino acid sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is directed. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, the preferred methods and materials are now described hereinafter. For convenience, the following definitions are also provided for use in describing the invention.
Term Definitions:
Growth factor: include both native and recombinant protein growth factors as well as biologically active fragments and analogs thereof capable of decreasing epidermal cell and thereby cutaneous senescence. These individual protein growth factors (epidermal growth factor (EGF), fibroblast growth factor (FGF) and transforming growth factor-alpha (TGF-alpha.) and fragments or analogs thereof are well known.
Collagen: the main protein component of bone, cartilage and other connective tissues; it is a microfibrillar protein whose subunit, tropocollagen, consisting of three polypeptide chains in which the amino acid sequence essentially (glycine-X—Y)_nwhere X and Y are often proline and hydroxyproline, respectively.
Epidermal cells: the outer epithelial portion of the skin, i.e. cuticle.
Cutaneous: is synonymous with the skin.
Skin: the membrane covering of a human body. The layers of the skin are the epidermis and the dermis.
Skin care product: the product is used to preserve health and youthfulness of human skin, or used to repair the wound and maintain integrity of human skin, include cosmetics and pharmaceuticals. The product can be applied for daily use or medical use; the medical use is in burn, scalding, trauma, or wound.
Effective amount to decrease senescence: the amount of fibroblast growth factor or composition thereof in a topical cosmetically or pharmaceutically acceptable carrier which is applied to human skin to achieve the desired result e.g., decrease cutaneous senescence in a human.
The complex structures of human skin are regulated by cytokines to maintain soft, smooth, and resilient skin. However, accompanying with aging, the synthesis of cytokines is decreasing or even imbalanced, this makes the skin dry and shriveled, loss resilience, and wrinkle appearance. The signaling factors are usually delivered between the skin cells including basic fibroblast growth factor (FGF-2 or bFGF) and kerationcyte growth factor (KGF or FGF-7). The main functions of FGF-2 include: regeneration of maturing tissues, synthesis of collagens and elastins, activation of fibroblast cells growth, activation of epidermal cells growth and move, synthesis of ureic acid, and wound healing. The mainly functions of KGF include: promotion of cell-cell tight connection, activation of cell proliferation and differentiation, activation of epidermal cells growth, and wound healing.
Another reason of cutaneous senescence is collagen loss. Collagen is a very important structural protein in animal, occupy 30% of total protein. The collagen in corium layer of human skin is broken by free radicals which is caused by ultraviolet (UV) light irradiation. The free radicals not only break collagens, but also damage the fibroblast cells, the collagen-producing cells. Further, accompanying with aging, the rate of collagen synthesis is slower than the rate of collagen degradation.
To decrease cutaneous senescence, collagen supplement is an important strategy used in common. However, the molecular weight of collagen is too large to pass through the epidermal layer of human skin; directly applying collagen on skin can't supply the lost collagen, unless the supplied collagen is processed specially. The safety of animal source of collagen is another concern, several co-infectious diseases between human and animals, such as bovine spongiform encephalopathy (BSE) or Japanese encephalitis in pig, and the residuals of antibiotics and hormones in economic animals is another problem.
The collagens are classified to 21 types. Depending on the amino acid sequence and composition of polypeptide chain, the different type of collagen is formed in different tissue. The collagen is produced by fibroblast cells. A prudent strategy for collagen supplement is providing growth factors to stimulate growth of collagen-producing fibroblast cells.
This invention provides a novel composition for promoting proliferation of fibroblast cells or epidermal cells comprising an effective amount of a fibroblast growth factor 1 (FGF-1).
The FGF-1 is an acidic fibroblast growth factor, and modulates the mitogenic activities of fibroblast cells growth. FGF-1 also stimulates proliferation of numerous types of cells and stimulates synthesis of collagens and elastins. FGF-1 represents strong capability of angiogenesis and applied for wound healing. A better embodiment is the composition comprising a human FGF-1 (hFGF-1), and the hFGF-1 includes wild type hFGF-1, recombinant hFGF-1, mutant hFGF-1, and derivatives thereof.

The wild type hFGF-1 has the following amino acid sequence:


(SEQ ID NO: 1)

	MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI

	LPDGTVDGTR DRSDQHIQLQ LSAESVGEVY IKSTETGQYL

	AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK

	NWFVGLKKNG SCKRGPRTHY GQKAILFLPL PVSSD.

Normally, post-translational modification of the wild type hFGF-1 removes the initiator amino acid methionine (M, shown in position 1 of SEQ ID NO: 1) from the N-terminus.

Providing hFGF-1 to human body is less concern of safety. Providing hFGF-1 to stimulate growth of fibroblast cells, further activate the collagen production of the fibroblast cells. In the preferred embodiment, the FGF-1 stimulates (1) the basic fibroblast growth factor signaling pathway to promote proliferation of fibroblast cells and (2) the keratinocyte growth factor signaling pathway to promote proliferation of epidermal cells.
Accordingly, the composition of the invention could be applied to produce skin care products. In the preferred embodiment, the skin care products comprises collagen, which have skin cells regeneration, stimulation of collagen synthesis, wrinkle elimination, senescence decrease, skin elasticity maintenance, wound healing, or vascularization. In the more preferred embodiment, the skin care products further comprises a cosmetically or pharmaceutically acceptable carrier. The products could be in the form of lotion, cream, mask, gel, emulsion, balsam, ointment, liquid, powder, or other types of topically or internally applicable material; and could be administered to a tissue such as intact skin, burn, scalding, and wound.
The hFGF-1 in this invention is characterized by a molecular weight of about 17.3 kDa when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and a pH value of about 7.0. The hFGF-1 stimulates not only fibroblast cell growth, but also epidermal cell growth. The proliferation of fibroblast cell and epidermal cell is mediated via the FGF-2 signaling pathway and the KGF signaling pathway, respectively. The proliferation of fibroblast cells is positive relates to collagen production in human body. The fibroblast cells proliferation by FGF-1 mediation can effectively supply collagen, maintain human skin elasticity, and decrease cutaneous senescence. Further, the proliferation of epidermal cells enhanced the skin cells regeneration.
The FGF-1 represents another capability of wound healing. FGF-1 is a potent angiogenic factors which induce formation of new capillary blood vessels. FGF-1 increases mesenchymal cell migration including smooth muscle cells and endothelial cells. Using FGF-1 for curing cutaneous trauma with a shortened recovering time in rodents is already known. The composition provided in this invention for increasing mesenchymal cell migration including smooth muscle cells and endothelial cells. This will promote tensile strength and rapid vascularization of wounds while accelerating the healing process. The composition is used for the treatment of wound healing or surgical healing of skin, vasculature, soft tissue or bone due to physical trauma, surgical procedures, fractures of bone or spinal cord, burns or soft tissue injury. The composition is also used for the treatment of tissue regeneration in areas of devascularized tissue or organ trauma, including central nervous system tissue and peripheral nervous tissue, vascularization of the myocardium after infarction.
This invention also provides a truncated hFGF-1, which has a deletion of amino acids No. 2 to No. 15. This type of hFGF-1 could be found in humans and is a product of proteolysis. The truncated hFGF-1 has the following amino acid sequence:

(SEQ ID NO: 2)

MFNLPPGNYK KPKLLYCSNG GHFLRILPDG TVDGTRDRSD

QHIQLQLSAE SVGEVYIKST ETGQYLAMDT DGLLYGSQTP

NEECLFLERL EENHYNTYIS KKHAEKNWFV GLKKNGSCKR

GPRTHYGQKA ILFLPLPVSS D.

This invention also provides hFGF-1 mutants for representing highly anti-oxidization properties comprising the following amino acid sequences:


(SEQ ID NO: 3)

	MAEGEITTFT ALTEKFNLPP GNYKKPKLLY CSNGGHFLRI
	LPDGTVDGTR DRSDQHIQLQ LSAESVGEVY IKSTETGQYL
	AMDTDGLLYG SQTPNEECLF LERLEENHYN TYISKKHAEK
	NWFVGLKKNG SSKRGPRTHY GQKAILFLPL PVSSD;
	and

(SEQ ID NO: 4)

	MAEGEITTFT ALTEKFNLPP GNYKKPKLLY SSNGGHFLRI
	LPDGTVDGTR DRSDQHIQLQ LSAESVGEVY IKSTETGQYL
	AMDTDGLLYG SQTPNEESLF LERLEENHYN TYISKKHAEK
	NWFVGLKKNG SSKRGPRTHY GQKAILFLPL PVSSD.

The mutants are prepared by substituting one or more cysteine residues in the amino acid sequence of the wild type hFGF-1 with serine. SEQ ID NO: 3 shows the substitution of cysteine 131 with serine, and is designated C131S. SEQ ID NO: 4 shows the substitutions of the three cysteines at positions 30, 97 and 131 with serine, and is designated CIIIS. The initiator methionine shown at position 1 of SEQ ID NO: 1 is normally removed during post-translational modification of the wild type hFGF-1 protein. Thus, alanine (A) is found at position 1 in the modified protein. When discussing cysteine residues at positions 30, 97, and 131 of the wild type hFGF-1 protein, the respective positions of those residues in SEQ ID NO: 1 are 31, 98, and 132. Similarly, the serine at position 131 of the hFGF mutant (C131S) refers to position 132 of SEQ ID NO: 3. Likewise, the serines at positions 30, 97, and 131 of the hFGF mutant (CIIIS) refer to positions 31, 98, and 132 of SEQ ID NO: 4.
The substitution of the cysteine residue with serine residue reduces the attack by the free radical and enhances the anti-oxidization ability, and represents longer half-life than wild type hFGF-1 dose; it means the mutant protein degrades more slowly than the wild-type protein. The mutant hFGF-1 further comprises the derivatives based on the SEQ ID NO: 3 and 4, or the derivatives based on the principle of substitution. The composition comprises the mutant hFGF-1 for skin care maintains a long-term effect for proliferation of fibroblast cells and epidermal cells.
Accordingly, the present invention provides a polypeptide for hFGF-1 comprises an amino acid sequence shown in SEQ ID NO: 4 (CIIIS) or its derivatives. SEQ ID NO: 4 (CIIIS) or its derivatives has all three cysteines in SEQ ID NO: 1 at positions 30, 97 and 131 being substituted with serine, and the polypeptide represents highly anti-oxidization properties.
The hFGF-1 including wild type, recombinant, mutant and derivatives are produced by the method comprising (a) producing a recombinant vector comprising hFGF-1 gene, (b) preparing a host cell for expressing the recombinant vector, and (c) purifying the hFGF-1 from the host cell culture with a heparin-sepharose column. The original source of hFGF-1 gene is from human, the gene is completed gene or partial gene expressing the FGF-1 functions. Then hFGF-1 is inserted into a recombinant vector, the vector is selected from a group consisting of plasmid, virus or phage vector. The recombinant vector expressing hFGF-1 is transformed or transfected into a suitable host cell such as bacteria, yeast, insect cell, or mammalian cell. Culturing the transformed/transfected host cell under suitable condition to express the hFGF-1 protein. Isolation and purification of the hFGF-1 is by well-known process such as affinity column. The affinity column is a heparin-sepharose column.
Based on the former descriptions, the composition provided in this invention comprises an FGF-1 that can be applied for skin care product. The skin care products are used in cosmetics or in medicine, including daily use for beauty treatment for the skin or the use for wound healing process. The composition further comprises a member selected from one group consisting of cosmetically acceptable carrier, or pharmaceutically acceptable carrier. The product type comprises liquid, semi-solid, and solid, such as lotion, cream, mask, gel, emulsion, balsam, ointment, liquid, powder, or other types of topically or internally applicable material.
Because a more youthful and pleasing appearance is the generally desired result of the method of the invention, the topical carrier is a topical cosmetically/pharmaceutically acceptable carrier. By “topical cosmetically/pharmaceutically acceptable carrier” as used herein is meant any substantially non-toxic carrier conventionally usable for topical administration of cosmetics or medicine in which the FGF-1 will remain stable and bioavailable when applied directly to the skin surface. For example, the FGF-1 can be dissolved in a liquid, dispersed or emulsified in a medium in a conventional manner to form a liquid preparation or is mixed with a semi-solid (gel) or solid carrier to form a paste, powder, ointment, cream, lotion or the like. Suitable cosmetically/pharmaceutically acceptable carriers are known to those of skill in the art and include cosmetically/pharmaceutically acceptable liquids, creams, oils, lotions, ointments, gels, or solids, such as conventional cosmetic night creams, foundation creams, suntan lotions, sunscreens, hand lotions, make-up and make-up bases, masks and the like. The composition can contain other ingredients effective in medicine, especially in wound healing, such as EGF, PDGF, or TGF-α. By the other way, the composition can contain other ingredients conventional in cosmetics including perfumes, estrogen, Vitamin A, C and E, alpha-hydroxy of alpha-keto acids such as pyruvic, lactic or glycolic acids, lanolin, vaseline, aloe vera, methyl or propyl paraben, pigments and the like.
Suitable topical cosmetically/pharmaceutically acceptable carriers include water, petroleum jelly (vaseline), petrolatum, mineral oil, vegetable oil, animal oil, organic and inorganic waxes, such as microcrystalline, paraffin and ozocerite wax, natural polymers, such as xanthanes, gelatin, cellulose, collagen, starch, or gum arabic, synthetic polymers, such as discussed below, alcohols, polyols, and the like. Preferably, because of its non-toxic topical properties, the carrier is a water miscible carrier composition that is substantially miscible in water. Such water miscible topical cosmetically/pharmaceutically acceptable carrier composition can include those made with one or more appropriate ingredients set forth above but can also include sustained or delayed release carrier, including water containing, water dispersable or water soluble compositions, such as liposomes, microsponges, microspheres or microcapsules, aqueous base ointments, water-in-oil or oil-in-water emulsions, gels or the like.
If desired, one or more additional ingredients conventionally found in topical cosmetic compositions can be included with the carrier: such as a moisturizer, humectants, odor modifier, buffer, pigment, preservative, vitamins such as A, C and E, emulsifier, dispersing agent, wetting agent, odor-modifying agent, gelling agents, stabilizer, propellant, antimicrobial agents, sunscreen, enzymes and the like. Those of skill in the art of topical cosmetic formulations can readily select the appropriate specific additional ingredients and amounts thereof.
This invention also provides methods for promoting proliferation of fibroblast cells or epidermal cells comprising administering an effective amount of a fibroblast growth factor 1, such as, for example, wild type hFGF-1, recombinant hFGF-1, mutant hFGF-1, derivatives thereof, or mixtures thereof. The fibroblast growth factor can have the amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, derivatives thereof, or mixtures thereof. This invention further provides methods for using fibroblast growth factor comprising administering an effective amount of one or more of the compositions as described herein to treat skin conditions. In some embodiments, this invention provides methods for regenerating skin cells, stimulating collagen synthesis, eliminating wrinkles, decreasing senescence, wound healing, maintaining elasticity, and/or synthesizing ureic acid, the methods comprising administering an effective amount of one or more of the compositions as described herein.

EXAMPLES

The following examples are offered by way of illustration and not by way of limitation.

Example 1

(A) Construction of Wild Type FGF-1 Expression Vector
The coding regions of human and bovine FGF-1 were amplified from existing cDNA clones by the polymerase chain reaction (PCR) using oligonucleotide primers that had restriction enzyme sites incorporated into them to facilitate cloning into the prokaryotic expression vector pET20b(+) (Novagen). The cloning, expression, and purification of newt FGF-1 is described elsewhere (Partie et al. Growth factor (1997) 14: 39-57). The human FGF-1 cDNA (Chiu et al. Oncogene (1990) 5: 755-762) was amplified with the sense primer BEF1 (5′-TGA AGC CAT ATG GCT GAA GGG GA-3′) (SEQ ID NO: 5) and the antisense primer HBGF605 (5′-AGA TCT CTT TAA TCA GAA GAG ACT-3′) (SEQ ID NO: 6). The bovine FGF-1 cDNA (Halley et al. Nucleic Acid Res. (1988) 16: 10913) was amplified with the sense primer BEF3 (5′-TGC TGA CAT ATG GCT GAA GGA GA-3′) (SEQ ID NO: 7) and the antisense primer BER3 (5′-CAA CAG ATC TCT TTA ATC AGA GGA GAC-3′) (SEQ ID NO: 8). PCR reactions were carried out in 10 mM Tris-HCl, pH 8.5, 50 mM KCl, 1.5 mM MgCl₂, 0.01% Tween 20, 0.01% Triton X-100, 0.25 mM dNTPs, 10 pmol of each primer, and 2 units of Taq polymerase. The reaction was initially denatured at 95° C. for 2 min followed by 30 cycles of denaturation at 95° C. for 1 min, annealing at 42° C. for 1 min (50° C. for human FGF-1), extension at 72° C. for 1 min, and finishing with a final extension at 72° C. for 7 min. The resulting 489 bp amplicon from the bovine FGF-1 cDNA template was gel purified, digested with NdeI and BgIII, and cloned into pET20b(+), which was previously digested with NdeI and BamHI, to yield the bovine FGF-1 expression vector, pETbovFGF-1. The gel-purified 485 bp amplicon from the human FGF-1 cDNA template was first cloned into pBluescript previously digested with SmaI. The resultant plasmid was then digested with NdeI and BgIII and the human FGF-1 cDNA insert cloned into pET20b(+) to yield the human FGF-1 expression vector pEThumFGF-1.
(B) Construction of Mutant FGF-1 Expression Vector
The amino acid at position 139 in the human FGF-1 sequence was mutated from a tyrosine to a phenylalanine (human Y139F), and the homologous amino acid in bovine and newt FGF-1 sequences was mutated from a phenylalanine to a tyrosine (bovine F139Y and newt F139Y) using the Transformer Site-Directed Mutagenesis Kit following the manufacturer's protocol (Clontech). The mutagenesis procedure (Myers et al. J Biol Chem (1995) 270: 8257-8266) was performed directly on the human, bovine, and newt FGF-1 expression vectors described above. The antisense selection primer used for the plasmid DNAs was 5′-GCA GCC ACT AGT AAC AGG ATT-3′ (SEQ ID NO: 9), which changes an AlwnI restriction site within pET20b (+) to an SpeI site. The antisense mutagenic primers used for the human, bovine, and newt FGF-1 sequences were 5′-CTT TCT GGC CAA AGT GAG TCC-3 (SEQ ID NO: 10)′, 5′-CTT TCT GGC CGT AGT GAG TCC-3′ (SEQ ID NO: 11), and 5′-CTT TTT GGC CAT AGT GGG TCC-3′ (SEQ ID NO: 12), respectively. The selection primer was also used in mutagenesis reactions for changing cysteine-131 of human FGF-1 to serine (human C131S) and serine-131 of bovine FGF-1 to cysteine (bovine S131C). The mutagenic primers used for the human C131S and bovine S131C sequences were 5′-GGA CCG CGT TTG GAG CTC CCA TTC-3′ (SEQ ID NO: 13) and 5′-GGA CCG AGT TTA CAC CTT CCG TTC-3′ (SEQ ID NO: 14), respectively. The mutated plasmids were verified by restriction enzyme analysis and direct sequencing of plasmid DNA.
The amino acid sequence of wild type hFGF-1 was shown in SEQ ID NO: 1. However, the hFGF-1 having the amino acid sequence shown in SEQ ID NO: 2 was found in humans. That was a truncated form of wild type hFGF-1 via proteolytic degradation. The amino acids No. 2-15 were deleted. SEQ ID NO: 3 shows the substitution of cysteine 131 of SEQ ID NO: 1 with serine, and is designated C131S. SEQ ID NO: 4 shows the substitutions of the three cysteines at positions 30, 97 and 131 with serine, and is designated CIIIS.

Example 2

Purification of FGF-1 Protein

The FGF-1 expression vectors were used to transform the bacterial expression host BL21(DE3)pLysS and then induced and purified as previously described (Partie et al. Growth factor (1997) 14: 39-57). In addition, 15 μL samples of the elution fractions from the heparin-Sepharose column were analyzed by 15% SDS-PAGE and silver staining to identify the protein-containing fractions and their purity. The first 29 amino acid residues of newt wildtype FGF-1 were determined using Protein Sequencer 475A (PE Applied Biosystems, Foster City, Calif.). The determined N-terminal amino acid sequence is identical to the predicted sequence saved the initiating methionine, which was cleaved following protein synthesis. We have also used matrix-assisted laser desorption ionization (MALDI) mass spectrometry to determine the molecular weights of the purified recombinant proteins. The LASERMAT 2000 Mass Analyzer (Finnigan MAT, San Jose, Calif.) was used for such measurements. The predicted molecular weight, observed molecular weight, and the percentage error are listed for human wildtype FGF-1 (17 330.0; 17 331.3; +0.008%), human mutant FGF-1 C131S (17 314.0; 17 311.2; −0.016%), and newt mutant FGF-1 F139Y (17 414.8; 17 410.3; −0.026%). Both protein sequencing analysis and molecular weight determination by mass spectrometry help to authenticate the identity of each recombinant protein. Bovine native FGF-1 (R&D Systems, Minneapolis, Minn.) represents a 140 amino acid form of bovine FGF-1 (amino acids 16-155).
The result is shown in FIG. 1. The purity of purified FGF-1 was achieved to 98%.

Example 3

Mitogen Assays

Swiss/3T3 cells were maintained in Dulbecco's-modified Eagle's medium (DMEM) supplemented with 10% calf serum and penicillin/streptomycin. Cells were seeded in 24-well plates at a density of 2×10⁴cells/well. At ˜80% confluency, the cells were washed once with phosphate-buffered saline (PBS) and placed in low-serum media (DMEM, 0.5% calf serum, penicillin/streptomycin) for 24 h. The cells were stimulated with recombinant human or recombinant bovine FGF-1 in the presence of heparin (10 μg/mL) for 20 h and then pulsed with 1 μCi of [3H] thymidine (NEN) for 6 h. As a positive control, calf serum at a final concentration of 10% was used. The labeling was terminated by washing the cells twice with 1 mL of PBS, twice with 1 mL of cold (4° C.) 5% trichloroacetic acid, and twice again with 1 mL of PBS. Cells were then solubilized in 0.8 mL of 0.25 M NaOH, and a 0.1 mL aliquot was counted in a Beckman scintillation counter.
The hFGF-1 is fully capable of eliciting a mitogenic response in Swiss/3T3 mouse fibroblasts comparable to that of the bovine recombinant FGF-1 used in these studies (FIG. 3). The optimal concentration of hFGF-1 used to induce mitogenic response is 10 ng/ml.

Example 4

FGF-1 Mediated Both FGF-2 and KGF Signaling Pathway

FGF1 mediated repression of skeletal muscle differentiation in MM14 cells expressing newt and human KGFR while FGF2 is not responsive to KGFR. KGF (also known as FGF-7) mediated repression of skeletal muscle differentiation in MM14 cells expressing newt and human KGFR. MM14 myoblasts were transfected with an α-cardiac actin/luciferase reporter plasmid, a CMV-LacZ plasmid, and the pBJ5 expression vector, the newt KGFR expression plasmid pNKGFR, or the human KGFR expression plasmid pHKGFR. The transfected cells were cultured either in the absence of any exogenously added growth factor or in the presence of FGF-1, FGF-2, or KGF. Cells were then harvested and assayed for luciferase and β-galactosidase activities. Reporter gene activity represents α-cardiac actin/luciferase activity of a given cell extract divided by the CMV/β-galactosidase activity measured for that extract.
The value obtained for each set of transfected cells cultured in the absence of growth factors was set at 1.0. The actual values of the pNKGFR and pHKGFR transfectants cultured with no growth factor were 72% and 62% that of the pBJ5 transfectant, respectively. It was showed that proliferating MM14 cells express KGF. The KGF that the MM14 cells were expressing may be sufficient to activate the transfected KGFRs and partially repressed activation of the reporter construct. The results shown in FIG. 3 were representative of three individual experiments.
It was shown that FGF-1 activated both KGF receptor and FGF-2 receptor. However, FGF-2 could not activate KGF receptor and the KGF couldn't activate FGF-2 receptor. FGF-1 could represent the effects of both FGF-2 and KGF.

Example 5

CuCl₂Treatment of Wild Type and Mutant FGF-1

Two micrograms of FGF-1 was incubated with Tris-HCl, pH 7.4, at a final concentration of 20 mM and CuCl₂at a final concentration of 10 mM in a total volume of 20 μL for 30 min at room temperature. The reaction was stopped by the addition of nonreducing 2× SDS sample buffer containing 10 mM EDTA and directly loaded on a 15% SDS-PAGE gel without boiling. The resulting gel was then subjected to Western blotting as above to visualize the proteins. For some experiments where protein samples were treated with CuCl₂, dithiothreitol was also added at a final concentration of 100 mM and the samples were boiled prior to SDS-PAGE and Western blotting.
The result was shown in FIG. 4. The capacity of wild-type human and bovine FGF-1 proteins as well as the human C131S and bovine S131C mutant FGF-1 proteins to form dimers was tested by treating them with CuCl₂, which has been previously shown to induce dimer formation of human FGF-1 (Engleka et al. J Biol Chem (1992) 267:11307-11315). As expected, wildtype human FGF-1 migrated with a size expected of monomers in the absence of CuCl₂, but in its presence there was a dramatic shift in the vast majority to a size corresponding to the size of dimers (FIG. 4, lanes 1 and 2). Interestingly, many fewer dimers were observed when the human C131S mutant was treated with CuCl₂(lanes 3 and 4), suggesting that cysteine-131 is responsible for the formation of most dimers induced by CuCl₂treatment. This, in turn, suggests that the two conservative cysteine residues, cysteine-30 and cysteine-97, do not contribute significantly in the dimer formation. Consistent with this observation, both the wild-type and S131C bovine FGF-1 proteins, which have an additional cysteine at position 61, form dimers in the presence of CuCl₂to a degree that is similar to that of wildtype human FGF-1 (lanes 5-8). Subsequent reduction of the CuCl₂-treated FGF-1 proteins with dithiothreitol showed that the dimer formation is a reversible reaction (lanes 9-12).

Claims

1. A composition for promoting proliferation of fibroblast cells or epidermal cells comprising an effective amount of a fibroblast growth factor 1 (FGF-1).

2. The composition of claim 1, wherein the FGF-1 is human FGF-1 (hFGF-1).

3. The composition of claim 2, wherein the hFGF-1 is selected from the group consisting of wild type hFGF-1, recombinant hFGF-1, mutant hFGF-1, and derivatives thereof.

4. The composition of claim 3, wherein the wild type hFGF-1 has the amino acid sequence shown in SEQ ID NO: 1.

5. The composition of claim 3, wherein the hFGF-1 has the amino acid sequence shown in SEQ ID NO: 2.

6. The composition of claim 3, wherein the hFGF-1 has the amino acid sequence shown in SEQ ID NO: 3.

7. The composition of claim 2, wherein the hFGF-1 is characterized by a molecular weight of about 17.3 kDa when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions.

8. The composition of claim 1, wherein the FGF-1 modulates the mitogenic activities of fibroblast cells.

9. The composition of claim 1, wherein the FGF-1 stimulates the basic fibroblast growth factor signaling pathway to promote proliferation of fibroblast cells.

10. The composition of claim 1, wherein the FGF-1 stimulates the keratinocyte growth factor signaling pathway to promote proliferation of epidermal cells.

11. The composition of claim 1, which applies to skin care products.

12. The composition of claim 11, wherein the skin care products comprises collagen.

13. The composition of claim 11, wherein the products have skin cells regeneration, stimulation of collagen synthesis, wrinkle elimination, senescence decrease, skin elasticity maintenance, wound healing, or vascularization.

14. The composition of claim 11, wherein the product further comprises a cosmetically or pharmaceutically acceptable carrier.

15. The composition of claim 11, wherein the product type comprises lotion, cream, mask, gel, emulsion, balsam, ointment, liquid, powder, or other types of topically or internally applicable material.

16. The composition of claim 13, wherein the product is administered to intact skin, burn, scalding, or wound.

17. The composition of claim 1, wherein the hFGF-1 is produced by the method comprising (a) producing a recombinant vector comprising hFGF-1 gene, (b) preparing a host cell for expressing the recombinant vector, and (c) purifying the hFGF-1 from the host cell culture.

18. The composition of claim 17, wherein the recombinant vector is selected from a group consisting of plasmid, virus and phage vector.

19. The composition of claim 17, wherein the host cell is selected from a group consisting of bacteria, yeast, insect cell, or mammalian cell.

20. A polypeptide for hFGF-1 comprises an amino acid sequence shown in SEQ ID NO: 4 or derivatives thereof.

21. The polypeptide of claim 20, which represents longer half-life than wild type hFGF-1 as shown in SEQ ID NO: 1.

22. The composition of claim 3, wherein the hFGF-1 has the amino acid sequence shown in SEQ ID NO: 4.