An epidermis‐permeable dipeptide is a potential cosmetic ingredient with partial agonist/antagonist activity toward fibroblast growth factor receptors
Ryuji Yamada MS1 | Riona Fukumoto BS2 | Chisato Noyama BS2 | Akio Fujisawa PhD2 |
Syuichi Oka PhD3 | Toru Imamura PhD1,2
1Cell Regulation Laboratory, Bionics Department, Tokyo University of Technology, Hachioji, Japan
2School of Bioscience and Biotechnology, Tokyo University of Technology, Hachioji, Japan
3Okinawa Life Science Research Center, Bio‐Sight Capital, Inc, Uruma, Japan
Correspondence
Toru Imamura, Cell Regulation Laboratory, Bionics Department, Tokyo University of Technology, 1404‐1 Katakura, Hachioji, Tokyo 192‐0982, Japan.
Email: [email protected]
Abstract
Background: Fibroblast growth factors (FGFs) are promising agents with which to treat problems of skin and hair. But their inability to penetrate into the skin due to their large size and hydrophilic nature prevents their topical application as effective cosmetic ingredients.
Aims: To identify small peptide(s) with FGF‐like activity and epidermis permeability. Methods: Several peptides deduced from our earlier studies were tested for their ability to promote keratinocyte growth and to activate FGF receptors (FGFRs). Permeability was assessed using HPLC after derivatization.
Results: A dipeptide, prolyl‐isoleucine (Pro‐Ile), not only stimulated growth of human keratinocytes, it also moderately activated FGFR3c and FGFR4, and activated FGFR1c to a lesser extent. This receptor specificity of Pro‐Ile is similar to that of FGF18. The activity of Pro‐Ile toward FGFR/BaF3 cells was enhanced by heparin and was inhibited by an FGFR inhibitor, PD173074. Pro‐Ile enhanced the activity of 5 ng/mL FGF18, but suppressed the activity of 50 ng/mL FGF18 toward FGFR3c and FGFR4. Pro‐Ile was found to permeate through validated model human epidermis. Conclusions: These results indicate that the dipeptide Pro‐Ile acts as a partial agonist/ antagonist for FGFR signaling, that it has receptor specificity similar to FGF18, and that it is able to penetrate into the model epidermis. Because FGFs expressed in the cutaneous system are physiological regulators, these results suggest the potential utility of this peptide as a topically applicable cosmetic ingredient for the regulation of skin physiology, hair growth, and wound healing.
K E Y WO R D S
dipeptide, epidermis, fibroblast growth factors, permeability, receptor
1 | INTRODUC TION
Fibroblast growth factors (FGFs)1 are key regulators of multiple phys‐ iological and pathological processes in various organs, including the skin. This makes them promising agents for use in cosmetic products.
Indeed, FGF2 is an approved drug used in the treatment of ulcerous wounds and burns,2 but is often administered subcutaneously for the off‐label use of facial rejuvenation and cosmetic surgery.3 Within the skin, several members of the FGF family are involved in physio‐ logical regulation of the hair growth cycle4‐7 and wound healing8,9
J Cosmet Dermatol. 2019;00:1–8. wileyonlinelibrary.com/journal/jocd
© 2019 Wiley Periodicals, Inc. | 1
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as well as in related pathologies. Subcutaneous administration of FGFs has been shown to stimulate/suppress hair growth10 and to enhance growth of fibroblasts that synthesize collagen, leading to reductions of facial skin wrinkles and nasolabial folds.11 Application of FGF2 to open skin sores dramatically accelerates wound heal‐ ing.8 Intravenous injection of FGF7 prevents and cures oral muco‐ sitis resulting from radiotherapy in cancer patients.12,13 FGF18 is a crucial regulator that maintains the resting phase of the hair growth cycle,4,7 and manipulation of its endogenous activity/expression could potentially enhance or suppress hair loss or growth. All of these effects suggest that FGF ligands can be utilized as versatile agents for repairing damaged/aged skin and for maintaining healthy and/or young skin. However, FGFs are hydrophilic polypeptides with molecular weights of 17‐25 kDa.14 They cannot efficiently penetrate into skin. In addition, repeated administration of large polypeptides could result in sensitization of host immune system. We therefore endeavored to identify small peptide(s) with FGF‐like activity and epidermis permeability.
2 | MATERIAL S AND METHODS
2.1 | Reagents
Peptides were obtained from the American Peptide Co. (Fisher Scientific). Recombinant FGF1 and FGFC (FGF Chimera),15 which are active with all FGF receptor (FGFR) subtypes, were prepared as described previously.15 Heparin and the FGFR inhibitor PD173074 were purchased from Sigma. Dansyl chloride (DC) was purchased from Tokyo Chemical Industry Co., Ltd. Other chemicals were purchased from Wako Pure Chemical Industries Ltd.
2.2 | HaCaT cell proliferation assay
HaCaT human epidermal keratinocytes were subcultured in calcium‐ free Dulbecco’s modified Eagle medium (DMEM; Gibco, 21068‐028) supplemented with 5% fetal bovine serum (FBS; HaCaT growth medium: final calcium ion concentration: approximately 0.03 mM). HaCaT cells were suspended in growth medium and plated into each well of a 96‐well plate at a density of 4 × 103 cells/100 µL and cultured for 24 hours at 37°C under 5% CO2 to allow the cells to attach. The cells were then rinsed, re‐fed with 100 µL of calcium‐ free DMEM supplemented with 0.03 mM calcium chloride and 0.1% FBS (HaCaT starvation medium) and cultured for 24 hours. The cells were then re‐fed with 100 µL of starvation medium containing the test samples at various concentrations and cultured for 45 hours. Thereafter, 10 µL of WST‐8 reagent (Cell Counting Kit‐8; Chemical Dojin Co., Ltd) was added to each well. After incubation for an additional 3 hours, the absorbance at 450 nm was measured using a microplate reader Multiskan FC (Thermo Scientific). By using this kit, highly water‐soluble tetrazolium salt, WST‐8, is reduced by dehydrogenase activities in cells to give a yellow‐color formazan dye, which is directly proportional to the number of living cells.
2.3 | BaF3 cell‐based FGFR activation assay
BaF3 cells (Riken RCB0805; Ba/F3) and WEHI‐3 cells (Riken RCB0035) were originally obtained from the RIKEN BioResource Center (Ibaraki, Japan). BaF3 cells are interleukin (IL)‐3 dependent pro B cell line without endogenous expression of FGF receptors. WEHI‐3 cells are lymphocyte‐like cells that had been established from murine leukemia. As WEHI‐3 cells secrete IL‐3, WEHI‐3‐ conditioned medium is used for the routine subculture of BaF3 cells. Construction of stable BaF3 cell transfectants, each overexpressing one of the FGFR subtypes modified with a common intracellular tyrosine kinase domain, and analysis of FGF‐induced cell growth were performed essentially as previously described.15 These cells have been extensively cloned and maintained based on their expression of FGF receptors and their responsiveness to FGFC, the universal FGFR ligand.15 The FGFR/BaF3 cells were routinely cultured using RPMI1640 medium supplemented with 10% FBS, 10 ng/mL FGFC, 5 µg/mL heparin, and 60 µg/ mL kanamycin. This culture condition ensures the presence of functional FGFR in each FGFR/BaF3 cells. Parent BaF3 cells were routinely cultured in RPMI1640 medium supplemented with 10% FBS, 10% WEHI‐3‐conditioned medium and 60 µg/ mL kanamycin (BaF3 growth medium) at 37°C under 5% CO2. FGFR‐overexpressing BaF3 transfectants were routinely cultured in RPMI1640 medium supplemented with 10% FBS, 20 ng/mL FGFC,15 5 µg/mL heparin, and 500 µg/mL G418 (growth medium) at 37°C under 5% CO2. For experiments, cells were washed once with RPMI1640 supplemented with 10% FBS (assay medium) and plated into 96‐well plates at a density of 2 × 104 cells/100 µL of assay medium/well. Peptide or FGF samples diluted in 11 µL of assay medium were added to each well and cultured for 45 hours at 37°C. When the effect of FGF1 was assessed, heparin was included at a final concentration of 5 µg/mL. Thereafter, 10 µL of WST‐8 reagent (Cell Counting Kit‐8; Dojin Chemicals) was added to each well. After incubation for an additional 3 hours, the absorbance at 450 nm was measured using a microplate reader.
2.4 | Epidermis permeability assay
A 3‐D model of human epidermis “LabCyte EPI‐MODEL” was purchased from the Japan Tissue Engineering Co. Ltd. This product was produced by culturing human epidermal cells, and the surface of this model epidermis were keratinized by exposure to the air. The similarity of this model to human epidermis has been confirmed by the manufacturer. For each culture insert, the surface of the epidermal layer was conditioned once and rinsed with 400 µL of DMEM. Each insert was then placed into a well of a 24‐well plate that was filled with 400 µL of medium. Pro‐Ile peptide solution (40 mM × 30 µL) was then applied to a paper disk (6 mm in diameter) that was placed on the cornified surface of the model epidermis, and the whole set was incubated for 12 hours at 37°C. The culture medium in the lower chamber was collected and assayed for permeated peptide.
2.5 | Detection and quantitation a fluorescent Pro‐ Ile derivative
To determine the concentration of permeated Pro‐Ile in the subepidermal compartment, fluorescence derivatization of Pro‐Ile and its measurement using an high‐performance liquid chromatography (HPLC) system equipped with a fluorescence detector (HPLC‐FL) were carried out. The fluorescent probe DC was dissolved in acetone (10 mM) and stored at room temperature until used. Tranexamic acid (TXA), which served as an internal standard (100 μM), was dissolved in 200 mM sodium carbonate buffer (pH 8.9). Authentic standard aqueous Pro‐Ile solutions (PI: 10, 20, 30, 40, and 50 μM) were also prepared for calibration. Equal aliquots of DC solution, TXA solution, Pro‐Ile solution, or reservoir solution containing the permeated peptide were incubated at 45°C for 2 hours.
2.6 | Analysis of the Pro‐Ile derivative using HPLC‐ FL
To isolate and measure the fluorescent Pro‐Ile derivative, isocratic HPLC‐FL analysis was performed using an Octa Decyl Silyl (C18) column (YMC, 5 mm, 150 × 4.6 mm) with a mobile phase composed of 50% methanolic water containing 0.2% formic acid delivered at 1.0 mL/min. For fluorescence measurements, the excitation wavelength was 325 nm and the emission wavelength was 525 nm.
3 | RESULTS
3.1 | Growth of human keratinocytes is stimulated by Pro‐Ile in a FGFR‐dependent manner
In our earlier study, we found that a pentapeptide, Gly‐Pro‐Ile‐Gly‐ Ser, stimulated proliferation of human hair keratinocytes and had a marked effect on human hair shaft elongation in organ culture.16,17 Here we found that the tetrapeptide Gly‐Pro‐Ile‐Gly (GPIG) stimulates HaCaT human keratinocytes (Figure 1A). To determine whether this activity is dependent on FGF signaling, the FGFR inhibitor PD173074 was added to the culture medium. As shown, PD173074 significantly inhibited the growth‐promoting activity of Gly‐Pro‐Ile‐Gly (Figure 1A). In an effort to identify smaller molecules potentially able to penetrate the epidermis, we assessed the ability of dipeptides comprising partial sequences of the tetrapeptide to stimulate HaCaT cell growth. We found that Pro‐Ile (PI, Figure 1B) and Ile‐Gly (IG, Figure 1C) each promoted HaCaT cell growth. However, only growth promoted by Pro‐ Ile was inhibited by PD173074 (Figure 1B and 1C). By themselves, the amino acids Pro (Figure 1D) and Ile (Figure 1E) did not exert significant growth‐promoting activity toward HaCaT cells.
3.2 | Pro‐Ile activates FGFR3c and FGFR4
The cell‐surface tyrosine kinase receptors for FGFs are encoded by four genes: FGFR1, FGFR2, FGFR3, and FGFR4. The transcripts of FGFR1‐3 are alternatively spliced to form two subtypes of
FGFR proteins for each of the three genes, and in each case, the splice variants exhibit independent ligand‐binding specificities.18 Consequently, there are seven major receptor subtypes for FGFs: FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b, and FGFR4.
Among these, FGFR1c, FGFR2b, FGFR3c, and FGFR4 are considered to be especially important in determining the physiological target of each FGF ligand. We therefore examined the activity of Pro‐Ile toward each of these FGFR subtype using a BaF3 cell‐based assay system for FGF signaling that has been successfully used in a number of our earlier studies.15,19 In these cells, the intracellular kinase domain of each FGFR was replaced with that of FGFR1 so that the mitogenic signaling could be compared between different FGFRs. We found that Pro‐Ile (PI) activates FGFR3c (Figure 2A) and FGFR4 (Figure 2B). In the figures, the extent of the FGFR activation is expressed as a percentage of that elicited by 25 ng/mL FGF1 in the presence of heparin. In that context, activation of FGFR3c and FGFR4 by Pro‐Ile was around 30% of that induced by FGF1. Inclusion of heparin enhanced the activity of the dipeptide, as is the case with many FGF proteins.20 Pro‐Ile also activated FGFR1c, but to a lesser degree (Figure 2C). By contrast, Pro‐ Ile did not significantly activate FGFR2b (Figure 2D). Pro‐Ile did not stimulate proliferation of the parent BaF3 cells that were used as the host cells for the overexpressed FGFRs (Figure 2E).
The activation of FGFR3c and FGFR4 by Pro‐Ile was inhibited by
PD173074, an inhibitor of the tyrosine kinase domain of these FGFR constructs (Figure 3A and 3). Taken together, these results confirm that Pro‐Ile is capable of activating some FGFR subtypes to exert its biological activity.
3.3 | Pro‐Ile is a partial agonist/antagonist for FGF18 signaling through FGFR3c and FGFR4
Because the receptor specificity of Pro‐Ile described above is reminiscent of FGF18, we tested whether Pro‐Ile might affect the activity of FGF18 toward these FGFRs. We found that Pro‐Ile weakly enhanced the activity of 5 ng/mL FGF18 toward FGFR3c, but it attenuated the activity of 50 ng/mL FGF18 (Figure 4A). Similarly, Pro‐Ile enhanced the activity of 5 ng/mL FGF18 toward FGFR4, but it attenuated the activity of 50 ng/mL FGF18 (Figure 4B). Pro‐Ile thus appears to be a partial FGF18 agonist/antagonist toward FGFR3c and FGFR4.
3.4 | Pro‐Ile is able to permeate through the epidermis in a 3‐D model of human epidermis
Topically applicable substances with FGF activity have long been sought. We therefore examined whether Pro‐Ile is able to permeate through the epidermis. Pro‐Ile solution was applied to the cornified surface of a 3‐D human epidermis model, incubated for 12 hours, and the reservoir medium that was in contact with the underside of the epidermis was collected. Aliquots of the collected reservoir solution and the applied solution were then analyzed for Pro‐Ile using HPLC‐FL after dansyl chloride (DC) derivatization (Pro‐Ile‐DC). Figure 5A shows a chromatogram for authentic PI‐DC standard.
F I G U R E 1 Effects of peptides and amino acids on the growth of HaCaT cells. Serum‐starved undifferentiated HaCaT cells were stimulated for 45 h with the indicated peptides or amino acids at the indicated concentrations. Cell numbers were evaluated by using WST‐8 reagent. In some cultures, the FGFR inhibitor PD173074 was included in the culture medium at 150 nM. A, B, Growth induced by the tetrapeptide Gly‐Pro‐Ile‐Gly (A) or dipeptide Pro‐Ile (B) and its suppression by the FGFR inhibitor. C, Growth induced by the dipeptide Ile‐Gly was unaffected by the FGFR inhibitor. D, E, Pro (D) and Ile (E) have no significant effect on growth. Symbols depict means ± SE of quintuplicate measurements. All the experiments were repeated twice with essentially the same results
TXA‐DC serving as an internal standard (IS) and Pro‐Ile‐DC as the
test sample were detected with chromatographic retention times of
14.8 and 29.0 minutes, respectively. The TXA‐DC and PI‐DC peaks were then confirmed to correspond to their molecular weights using an HPLC system equipped for time‐of‐flight mass spectrometry (TOFMS) (data not shown). A calibration curve showed good correlation between the ratio of the areas under the Pro‐Ile and TXA peaks (Pro‐Ile/TXA) and the Pro‐Ile concentration (Figure 5B). Pro‐ Ile was also detected in samples of the reservoir solution (Figure 5C) but not in the blank (Figure 5D), which indicates that the Pro‐Ile was able to permeate through the epidermis in this model. Using the calibration curve, the concentration of Pro‐Ile in the reservoir solution was determined to be 10.5 μM. The ratio of permeated amount/applied amount of Pro‐Ile in this experiment was calculated to be 0.35%.
4 | DISCUSSION
Unmet needs for agents that modify the behavior of cells in cutaneous systems are growing. Especially sought are agents which when applied topically to the skin surface will effectively reach regions of interest, including the epidermis, dermis, hair follicles, and other structures, to reverse aging phenomena affecting the skin and hair. FGFs involved in the physiological and/or pathological regulation of cutaneous systems are among the agents thought to have therapeutic potential. However, FGFs are hydrophilic polypeptides with molecular masses ranging between 17 kDa and 25 kDa14 and are not easily deliverable into skin via topical application. Thus, our findings that a dipeptide, Pro‐Ile, acts as a partial FGFR agonist/antagonist and that Pro‐Ile is able to permeate through the epidermis are very promising and suggest this peptide may be a useful cosmetic ingredient.
F I G U R E 2 Activation of FGF receptors FGFRs by Pro‐Ile. BaF3 cell transfectants stably overexpressing one FGFR subtype were stimulated with Pro‐Ile at the indicated concentrations for 45 h at
37°C. Control cells were transfected with empty vector. For some cells, heparin was included (5 µg/mL) in the culture medium. Cell numbers were evaluated using WST‐8 A, FGFR3c/BaF3 cells. B, FGFR4/BaF3 cells. C, FGFR1c/BaF3 cells. D, FGFR2b/BaF3 cells. E, Parent BaF3 cells without endogenous FGFR. Symbols depict means ± SE of quintuplicate measurements. All the experiments were repeated twice (except 3× for figure E) with essentially the same results
F I G U R E 4 Pro‐Ile is a partial agonist/antagonist of FGF18 signaling through FGFR3c and FGFR4. BaF3 cell transfectants stably overexpressing FGFR3c (A) or FGFR4 (B) were stimulated with FGF18 at 5 ng/mL or 50 ng/mL together with Pro‐Ile at the indicated concentrations for 45 h at 37°C. Cell numbers were evaluated using WST‐8 reagent. Symbols depict means ± SE of quintuplicate measurements. All the experiments were repeated three times with essentially the same results
F I G U R E 5 Pro‐Ile permeated through the epidermis in 3‐D model of human epidermis. Pro‐Ile was applied to the cornified (upper) surface of model epidermis and incubated for 12 h at 37˚C. The opposite (lower) surface of the model epidermis was immersed in assay medium. The Pro‐Ile content in the assay medium was detected by derivatization of Pro‐Ile using dansyl chloride (DC) followed by high‐performance liquid chromatography using a C18 reverse
phase column. Tranexamic acid (TXA) was
0 5 10 15 20 25 30 35 0 10 20 3040 50
also derivatized with DC and served as anRetention time (m(C) (D)
Pro-Ile concentration (µM)
internal standard (IS). A, Chromatogram of authentic Pro‐Ile‐DC standard. TXA‐ DC was analyzed as an internal standard (IS). B, Calibration curve showing good correlation between ratios of the areas
TXA-DC (IS) TXA-DC (IS)
Pro-Ile-DC
under the Pro‐Ile and TXA peaks (Pro‐Ile/ TXA) and the Pro‐Ile concentrations. C, Detection of permeated Pro‐Ile (Pro‐Ile‐ DC) in the assay solution. D, No (Pro‐Ile‐ DC) was detected in the blank 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Retention time (m) Retention time (m)
FGFR signaling,22 and identify FGF‐like activity in placental ex‐ tract for cosmetics.19
The growth‐promoting activity of Pro‐Ile and Gly‐Pro‐Ile‐Gly toward HaCaT cells had not initially been considered in the context of FGFR signaling, but we have now clearly demonstrated that this activity is FGFR‐dependent, as it was inhibited by the FGFR inhib‐ itor PD173074 (Figure 1A and 1). The results of several experi‐ ments examining FGFR activation using the FGFR/BaF3 cell‐based assay confirm this interpretation. The finding that, by themselves, neither Pro nor Ile promoted HaCaT cell proliferation indicates
that the activity of Pro‐Ile requires the structure of this dipep‐ tide (Figure 1D and 1), though the mechanism by which this pep‐ tide activates FGFRs is currently unknown. The Pro‐Ile sequence is found in the primary structure of human FGF5 (242Pro243Ile: NM_004464) and mouse FGF5 (41Pro42Ile and 242Pro243Ile: M37825), but both the 41Pro42Ile and 242Pro243Ile regions of FGF5 are located outside the “core” structure conserved among FGF family members. This suggests these Pro‐Ile sequences may mod‐ ulate the binding of FGF5 to FGFR, but are not primary mediators of the binding. Although the Pro‐Ile sequence is not present in the primary structure of FGF18, the Pro‐Ile dipeptide exhibits FGF18‐ like, not FGF5‐like, receptor specificity (described below). This suggests the presence of the Pro‐Ile sequence in FGF5 may be a mere coincidence. The mechanism by which heparin enhances the activity of Pro‐Ile is also of great interest. In the formation of the FGF‐FGFR complex, heparin directly binds to both the FGF ligand and the FGFR, establishing a tight molecular complex composed of FGF, FGFR, and heparin/heparan sulfate.23 Pro‐Ile, however, does not bind to heparin. Instead, heparin’s interaction with FGFR may enhance a Pro‐Ile‐induced conformational change in FGFR that is required for activation. Confirmation of this idea awaits further structural analysis.
Pro‐Ile dipeptide moderately activates FGFR3c and FGFR4 and more weakly activates FGFR1c. This is noteworthy because this specificity profile is similar to that of FGF18. Within the skin, FGF18 is selectively expressed in the hair follicle and epidermal stem cell niches, where it regulates stem cell activation. Hair fol‐ licle stem cells express FGF18 during the telogen phase of the hair cycle, maintaining the quiescent state of the stem cells and their daughter cells.4,7 For the hair follicles to enter a new growth phase (anagen), FGF18 expression/activity must be suppressed. Thus, if Pro‐Ile acts toward hair follicle stem cells through its bi‐ phasic activity toward FGFR3c and FGFR4, it may stimulate hair growth during telogen, but suppress hair growth during anagen. The permeability of the epidermis to Pro‐Ile would help the di‐ peptide to exert these hypothetical activities at the desired sites of action. On the other hand, FGFR1c is also stimulated by Pro‐Ile, although less strongly. FGFR1c signaling is a dominant activator of an ERK1/2 signaling pathway that leads to cellular proliferation. In that context, Pro‐Ile may enhance wound healing by stimulat‐ ing growth of epidermis and dermis. Thus, Pro‐Ile may have the potential to modulate several physiological events depending on spaciotemporal expression profile of the FGFR in the tissue.
In sum, our results indicate that the dipeptide Pro‐Ile is able to
penetrate the epidermis and that it acts as a partial agonist/antag‐ onist for FGFR signaling with receptor specificity similar to FGF18. Because FGFs are expressed in cutaneous systems (eg, hair folli‐ cles) and act as physiological regulators, these results suggest the potential utility of this peptide as a topically applicable cosmetic ingredient for regulating skin physiology, hair growth, and wound healing.
ACKNOWLEDG MENTS
We thank Prof. Jin Masaki at Tokyo University of Technology for his technical advice on the permeability experiment. We also thank Mss. Yukari Arita and Nobuko Ito at KINKANDO CO. LTD. for discussion. This manuscript was edited by Dr. William Goldman at MST Editing Co.
ORCID
Toru Imamura https://orcid.org/0000‐0001‐7331‐6004
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