Control of Hair Growth with Parathyroid Hormone (7-34)
Marcus B. Schilli,*t Swapna Ray,* RalfPaus,t Eliot Obi-Tabot,* and Michael F. Holick* * Vitamin D, Skill and Bone Research Laboratory, Endocrine Section, Department of Medicine, Boston University Medical Center, Boston, Massachusetts, U.S.A.; and ^Department of Dermatology, Charite Hospital, Humboldt-Universitat. Berlin, Germany
Parathyroid hormone (PTH) related peptide (PTHrP) is thought to influence the proliferation and differentiation of the epidermis and hair follicle. As a means of elucidating the biologic function of PTHrP on the hair follicle, a PTHrP analog PTH (7-34), which is a PTH/PTHrP receptor antagonist, was given intraperitoneally twice daily to C57 BL/6 mice at different stages of the hair cycle. PTH (7-34) induced 99 ± 4.5% (mean ± SEM) of resting telogen hair follicles into a proliferative (anagen) state, whereas 100% of the hair follicles in the control group remained in telogen. To determine whether this peptide influenced the progression of the hair follicles from anagen to catagen (hair follicle maturation and regression), groups of mice that were either spontaneously in or induced to anagen received either PTH (7-34) or placebo. Morphometric analysis of the hair follicles from the middle back region of the spontaneous anagen mice that received PTH (7-34) revealed that 19 ± 4% (mean ± SEM) of the follicles were in anagen VI, whereas none (0%) were in anagen in the control group. Similarly, in induced anagen mice treated with PTH (7-34), 22.3 ± 1.4 (mean ± SEM) of the follicles were in anagen VI compared to only 1.3 ± 0.7% in the control mice. Together these observations suggest that PTHrP is a hair follicle morphogen that may be a major factor responsible for controlling the hair cycle. These studies provide a new insight for development of PTHrP analogs for a wide variety of disorders related to disturbances of hair cycling. Key words: parathyroid hormone related peptide/mice/hair cycle/anagen. J Invest Dermatol 108:928-932, 1997 Parathyroid hormone (PTH)-related peptide (PTHrP) is a 141-amino acid protein that shares 70% homology with PTH in its first 13 amino acids but diverges completely in its primary structure thereafter (Broadus et al, 1988; Thiede et al, 1988; Orloffrt al, 1994; Martin et al, 1991). Studies using synthetic PTH and PTHrP amino-terminal fragments have demonstrated that these peptides bind to the PTH receptor and cause biologic effects on calcium and phosphorus metabolism. A PTH/PTHrP receptor has been cloned, and it has been demonstrated that PTH/PTHrP mRNAs are widely expressed in many tissues unrelated to calcium metabolism, including the skin (Juppner et al, 1991; Urena et al, 1993). PTHrP is similarly expressed in several normal and neoplastic tissues, including skin and hair follicles (Merendino et al, 1986; Hayman et al, 1989; Arillasoy, 1991). Although the physiologic role of PTHrP in the skin is not well understood, mounting evidence suggests that this peptide plays an important role in epidermal proliferation and differentiation1,2 (Henderson et al, 1992; Kaiser et al, 1992). The PTH/PTHrP receptor agonist PTH (1-34) was found to inhibit epidermal proliferation in cultured human keratinocytes (Holick et al, 1994), and in vivo in SKH-I hairless mice (Holick et al, 1994). Evidence that this effect was mediated through the PTH/PTHrP receptor was provided by studies in which the addition of the PTH/PTHrP receptor antagonist, PTH (7-34) reversed the PTH (l-34)-induced anti-proliferative effect on cultured keratinocytes1 (Holick et al, 1994). In vivo PTH (7-34) stimulated, in a dose-dependent fashion, [3H]thymidine incorporation into the epidermis. Furthermore, PTH (7-34) increased the total number of visible hair shafts and hair length by 146% and 80%, respectively (Holick et al, 1994). To further investigate the biologic function of PTHrP as a possible regulator of hair growth, we investigated the effect of its antagonist PTH (7—3 4) on the cyclic growth and regression of hair follicles in mice with normal hair growth using the C57 BL/6 mouse model for hair research (Paus et al, 1990, 1994a, 1994b, 1994c).
MATERIALS AND METHODS
C57 BL/6 mice were purchased from Charles River Breeding Laboratories (Wilmington, MA) and fed a standard mouse chow diet ad libitum. [Nle8,18Tyr34] parathyroid hormone (7-34)-amide [PTH (7—34)] was purchased from Bachem California (Torrance, CA). Two groups of six to seven telogen 24-d-old C57 BL/6 mice were given either 1 µg of PTH (7-34) in 0.1 ml of distilled water or 0.1 ml of distilled water twice daily interperitoneally for 14 d. The skin color for the mice in each group was evaluated on day 12, 13, and 14 of treatment as previously described (Paus et al, 1990)
Manuscript received April 8, 1996; revised February 18, 1997; accepted for publication February 28, 1997. Reprint requests to: Michael F. Holick, Ph.D.. M.D., 80 East Concord Street, Boston University Medical Center, M1013, Boston, MA 02118. Abbreviations: PTH, parathyroid hormone; PTHrP, parathyroid hormone related peptide, PTH (7—34), parathyroid hormone (7—34). 1 Holick MF, Nussbaum S, Persons KS: PTH-like humoral hypercalcemia factor (HHF) of malignancy may be an epidermal differentiation factor: synthetic hHHF(l—34)NH^ inhibits proliferation and induces terminal differentiation of cultured human keratmocytes.J Bone Miner Res 3:S214, 1988 (abstr). 2 Milstone L, Fairley J, Insogna K, Stewart A. Reuveni H: Parathyroid hormone-like peptides increase intracellular calcium and differentiation of keratinocytes.J Cell Biol 107:73A, 1988 (abstr.)
On day 14 of treatment, the mice were sacrificed for histologic and histomorphometric studies. The mice were sacrificed on day 14, and the whole back skin was harvested. Defined regions (neck, middle, and tail area of the back) were obtained following a standardized protocol (Paus et al, 1990, 1994a, 1994c). After histologic processing (fixation, paraffin embedding, Geimsa staining), the morphologic analysis and photography of the longitudinal sections of full-thickness skin were performed at 100 and 400 X magnification. For each of the test and control mice, at least 50 microscopic fields (10 X) were assessed per region of the back by morphometry. The data were pooled, and the means SEM and levels of significance were calculated using Mann-Whitney U-tests. The middle back region of all experiments was selected for this representation because it was the area with the most substantial changes.
To evaluate the effect of PTH (7-3 4) on inhibiting the progression of anagen into catagen, two groups containing seven 28-d-old anagen mice were given either 1 µg of PTH (7-34) in 0.1 ml of distilled water or 0.1 ml of distilled water twice daily interperitoneally for 9 d. Analysis of skin color, skin histology, and histomorphometry were conducted as described above.
To determine whether PTH (7-34) could inhibit anagen-induced maturation of the hair follicle, three groups of between four to six 6- to 8-wk-old telogen mice were depilated with a wax rosin. Starting on day 9 postdepilation, when all mice had reached anagen, the mice were given either 1µg PTH (7-34) or vehicle twice daily interperitoneally for 10 d. The skin color, skin histology, and skin histomorphometry were evaluated as described above. Serum calcium measurements were performed by our clinical laboratory using an automated system.
RESULTS
Acceleration of Anagen by PTH (7-34) in 24-d-old Mice That Were in Telogen We reasoned that if PTHrP is a factor that regulates hair growth, then a PTH/PTHrP receptor antagonist [PTH (7-34)] could block the inhibitory activity of endogenous PTHrP on the hair cycle. This would result in stimulation of the hair follicle from its telogen resting state into the proliferative anagen state, causing a prolongation of the duration of hair growth. We used the C57 BL/6 mouse, which has a highly synchronized hair cycle that can be easily evaluated because the hair follicle is the only location for melanin production, and melanin is only produced during active hair growth (Fig 1A) (Straile et al, 1961; Paus et al, 1990, 1994a, 1994c). Thus, skin color reliably indicated the different stages of the hair cycle.
We studied infantile 24-d-old C57BL/6 mice to determine whether the impending spontaneous initiation of new hair growth (anagen wave) could be further accelerated by PTH (7-34). There was a marked increase in the amount of pigmentation on the infantile mice receiving PTH (7-34), indicating that their hair follicles had been prematurely induced into anagen (Fig 1B). A macroscopic evaluation of the back skin from the mice receiving PTH (7-34) revealed a time-dependent increase in the skin area showing progression from telogen into anagen (Fig 1A). By the 14 day of treatment with PTH (7-34), there was a 131% increase in the area on the back that was in anagen when compared to the control mice. A representative histologic cross-section of the middle region of the back skin from a control mouse at day 14 of treatment showed the hair follicles were still in telogen (Fig 3A) whereas, in contrast, the hair follicles of mice that had received PTH (7-34) were in anagen (hair follicles producing melanin and hair shafts) (Fig 3B). Morphometric analysis of parallel reference areas in the middle back region revealed that after 14 days of treatment, the telogen animals that received PTH (7-34) showed 99 ± 4.5% (mean ± SEM) of the hair follicles in anagen, whereas 100% of the hair follicles in the control group remained in telogen (Fig 4A).
Prolongation of Anagen and Delay of Catagen by PTH (7-34) in Mice that Were in Spontaneous Anagen We next determined whether we could prolong anagen and delay catagen development in mice that were in spontaneous anagen with PTH (7-34). Mice that received PTH (7-34) showed a significant prolongation of anagen as judged by the larger area of dark black skin over the back when compared to controls (Fig 1C). The percent of back skin area in catagen and telogen progressed more rapidly in the control mice than in the PTH (7-34)-treated mice (Fig 2B). By day 9 of treatment, most of the back skin area of control mice were in catagen or telogen (67 ± 9.2%; mean ± SEM), whereas PTH (7-34) inhibited the progression into catagen (only 27.9 ± 6.0%; mean ± SEM of the back area of test mice were in catagen). This was quantified and confirmed by morphometric analysis (Fig 4B), which showed that 19 ± 4% (mean ± SEM) of the hair follicles in the middle back region remained in anagen VI in mice treated with PTH (7-3 4), whereas none (0%) were in anagen in the control group (Fig 4B). Furthermore, whereas almost 100% of the hair follicles of control mice progressed into late catagen and telogen, only 17 ± 9% (mean ± SEM) of the hair follicles had progressed into late catagen, and none of the follicles progressed to telogen in the PTH (7-34)-treated mice.
Inhibition of Catagen in Anagen-induced Mice by PTH (7-34) In the third set of experiments, 6- to 8-wk-old adolescent C57BL/6 mice with all follicles arrested in telogen for several weeks were selected. Anagen was induced in all back skin follicles by hair shaft depilation with wax/rosin-mixture (Paus et al, 1990, 1994a, 1994c). This achieves the highest degree of cycling synchrony obtainable by any technique. Seventeen to 19 d after anagen induction by depilation, these adolescent mice enter catagen spontaneously (Paus et al, 1990, 1994a, 1994c). We were interested in determining whether PTH (7-34) could retard spontaneous catagen development in these anagen-induced adolescent mice. This allowed us to judge, precisely, in a very large homogeneous population of anagen VI follicles, the effects of PTH (7-34) on various catagen-associated regression phases of mature hair follicles, which are characterized by coordinated keratinocyte differentiation and apoptosis (Straile et al, 1961; Paus et al, 1990, 1994a, 1994c).
Figure 1D shows representative animals just before sacrifice. The control mice had grayish-pink skin, especially in the neck area, which indicated the progression of the follicles in the hair cycle from catagen into telogen. In contrast, the mice that received PTH (7-34) had a substantially larger area of darker skin, consistent with the hair follicles remaining in anagen (Fig 1D). By the ninth day, PTH (7-3 4) markedly inhibited the progression of the hair follicle from anagen to catagen, and this effect was sustained to the 11th day (Fig 2C). Microscopic analysis of the hair follicles from mice treated with PTH (7-34) showed that 22.3 ± 1.4% (mean ± SEM) remained in anagen VI compared to only 1.3 ± 0.7% (mean ± SEM) of the control group (Figs 3C,D; 4C). The inhibition of catagen development by PTH (7-34) was further supported by the observation that the percentage of hair follicles in the early stages of catagen development was significantly higher in PTH (7-34)-treated mice than in the controls, where middle and late stages of catagen development dominated (Fig 4C).
Blood Calcium Determinations in the Control and PTH (7-34)-Treated Mice An evaluation of the blood from the control and treated mice for all experiments did not reveal any significant differences in the levels of calcium (8.7 ± 0.1 vs 8.6 ± 0.6; p = 0.84 in control and PTH (7-34)-treated mice, respectively).
DISCUSSION
Our findings provide convincing evidence that PTH (7-34), either by preventing endogenous PTHrP from interacting with its receptor (Juppner et al, 1991; Urena et al, 1993), by interacting with another as yet unidentified PTHrP receptor, or by affecting another unrelated receptor, accelerated anagen development in telogen follicles. In addition, PTH (7-34) maintained active hair follicle growth and hair shaft formation for a significantly longer period than found naturally. With the exception of FGF-5 (Hebert et al, 1994), this suggests that PTHrP is the only other endogenous factor identified to date to be critically involved in anagen termination or catagen induction, two currently indistinguishable and probably identical processes (Straile et al, 1961; Paus et al, 1991, 1994a, 1994b, 1994c).
The anagen induction and catagen inhibition in rapidly cycling infantile mice by PTH (7-34) may reflect an acceleration of the unknown "biologic clock" that drives hair cycling, PTH (7-34) could either be replacing the natural anagen initiation signal or counteracting PTHrP as a "brake" on anagen development. Alternatively, PTH (7-34) may directly antagonize the natural catagen initiation signal(s). That PTH (7-34) prolonged anagen and inhibited catagen in adolescent anagen-induced mice further supports the concept that PTHrP is a key signal for hair follicle regression because the follicles in these older mice were relatively slow cycling, which makes interference with the intrinsic generator of follicle cycling a less likely explanation. During its development and its postnatal cyclic remodeling, the hair follicle is essentially an epithelial-mesenchymal interactive system whose development depends on a tightly choreographed series of messages exchanged between the dermal and epidermal cells (Paus et al, 1990, 1991, 1994a, 1994b, 1994c). It has been hypothesized that the hair cycle is controlled by an inhibition/disinhibition system whereby a locally generated mitotic inhibitor ("chalone") that gradually accumulates during anagen causes entry of the follicle into catagen when present in sufficient concentrations (Chase, 1954). This "chalone hypothesis" further stipulates that the gradual loss of activity or the dispersal of the inhibitor during telogen eventually disinhibits follicle growth, a process that may be accelerated by the depilation of telogen hair shafts. It is now recognized that while human cultured keratinocytes and keratino-cytes surrounding the hair follicle produce PTHrP (Merendino et al, 1986;
Hayman et al, 1989), they do not possess a PTH/PTHrP receptor (Hanafin et al, 1995; Lee et al, 1995). It is not known whether the recently identified PTH2 receptor, which is found in the brain and pancreas, is present in the hair follicle or whether it recognizes PTH (7-34) (Usdin et al, 1995). Cultured human skin fibroblasts and the dermal papilla, however, express mRNA for the PTH/PTHrP receptor (Hanafin et al, 1995; Lee et al, 1995). Thus, if the hair follicle keratinocyte continues to produce PTHrP during its proliferative phase, it, in turn, could exert its antimitotic activity on the dermal papilla, thereby terminating further hair growth and initiating the follicle into catagen and telogen. This effect would render PTHrP an ideal candidate for being one of the long elusive cutaneous chalones (Chase, 1954; Paus et al, 1990, 1991, 1994b, 1994c).
There is additional evidence that PTHrP can alter the hair cycle. Transgenic mice that overexpressed PTHrP displayed a profound disturbance in hair follicle development, particularly in abdominal skin, where a complete lack of hair follicle formation was observed (Wysolmerski et al, 1994). It was also found that whereas the PTHrP agonist PTHrP (1-34) inhibited epidermal proliferation and had no effect on the number or length of hairs of SKH-1 mice, PTH (7-34) had a profound effect on enhancing epidermal proliferation and increasing the number and length of hair shafts (Holick et al, 1994). Because the SKH-1 hairless mouse is not a good model for hair research, however, it is difficult to compare those results with our observations in C57 BL/6 mice. Although it may be possible that PTH (7-34) had a direct effect on melanogenesis, there is no evidence from in vivo studies to suggest that PTHrP or PTH (7-34) alter melanogenesis (Holick et al, 1994; Wysolmerski et al, 1994). On the background of these data, our observations strongly suggest that PTHrP is a powerful hair cycle modulator involved in anagen termination and telogen maintenance that functions as a molecular brake on anagen.
Our observations demonstrating the dramatic effect of PTH (7—34) on the hair cycle offers a new opportunity to develop PTH analogs for treating disorders of hair growth. Although the PTH (7-34) may have a relatively short circulating half-life (Kukreja et al, 1994), it appears that once it reaches the hair follicle, its biologic half-life is prolonged, resulting in a profound effect on the hair cycle.
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