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).
