Protection against cyclophosphamide-induced alopecia and inhibition of mammary tumor growth by topical 1,25-dihydroxyvitamin D3 in mice.

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)2D3 was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)2D3, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)2D3-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)2D3 alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)2D3 pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)2D3 can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)2D3 was shown, either alone or in combination with CTX, to inhibit tumor growth.

Evidence that activation of protein kinase A inhibits human hair follicle growth and hair fibre production in organ culture and DNA synthesis in human and mouse hair follicle organ culture.

We have investigated the possibility that protein kinase A (PKA) may play a part in regulating the activity of human and mouse hair follicles in whole organ culture. Human hair follicles were isolated from facial skin by microdissection, and hair follicle and hair fibre length measurements were made daily during suspension culture. Incubation of human hair follicles with dibutyryl-cAMP (db-cAMP) resulted in a dose-dependent inhibition of total cumulative follicle growth (IC50 = 100 mumol/L, 85% inhibition at 1 mmol/L). db-cAMP (0.5 mmol/L) also caused a rapid, partial inhibition of follicular DNA synthesis (20.3% inhibition at 6 h, 48.0% inhibition at 24 h). Human hair follicle growth was inhibited by the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and Ro 20-1724, and by the adenylate cyclase activator, forskolin. In addition, db-cAMP inhibited DNA synthesis in organ cultures of whisker follicles isolated from neonatal mice by microdissection. Taken together, these findings indicate that agents which increase cAMP levels are potent inhibitors of human and mouse hair follicle growth, and suggest that PKA may play a part in the regulation of hair follicle activity in vivo.

Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

Regulation of human dermal papilla cell production of insulin-like growth factor binding protein-3 by retinoic acid, glucocorticoids, and insulin-like growth factor-1.

Insulin-like growth factor-1 (IGF1) has been reported to stimulate hair elongation and to facilitate maintenance of the hair follicle in anagen phase. However, little is known about IGF1 signaling in the hair follicle. In this study we investigate the effects of IGF1, glucocorticoids, and retinoids on dermal papilla (DP) cell production of insulin-like growth factor binding proteins (IGFBPs). IGFBPs comprise a family of IGF binding proteins that are produced and released by most cell types. They bind to IGFs to either enhance or inhibit IGF activity. In the present report we identify IGFBP-3 as being produced and released by cultured human dermal papilla (DP) cells. IGFBP-3 levels are increased fivefold by retinoic acid, eightfold by dexamethasone, and tenfold by IGF1. DP cells are known to produce IGF1, and so the observed stimulation of DP cell IGFBP-3 production by IGF1 is consistent with the idea that DP cells possess the IGF transmembrane receptor kinase and are autoregulated by IGFs. The level of another IGFBP, tentatively identified as IGFBP-2, is, in contrast, not regulated by these agents. IGFBP-3 has been shown to inhibit the activity of IGFs in a variety of systems. Our results are consistent with a model in which retinoids and glucocorticoids inhibit IGF action on DP cells and surrounding matrix cells by stimulating increased DP cell production of IGFBP-3. The IGFBP-3, in turn, forms a complex with free IGF1 to reduce the concentration of IGF1 available to stimulate hair elongation and maintenance of anagen phase.

Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture.

In this study we have used a human hair follicle whole-organ culture system to examine the effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), on hair follicle growth and hair fibre production. Anagen hair follicles were isolated from human facial skin by microdissection and placed in suspension culture in supplemented Williams E medium. Hair follicle and hair fibre lengths were measured daily using an inverted microscope and cumulative growth values were calculated. Treatment with TPA resulted in a potent, dose-dependent inhibition of total cumulative hair follicle growth (IC50 = 1 nM). Hair follicles grew at a comparable rate for 4 days in the presence or absence of 10 nM TPA, after which growth of TPA-treated follicles ceased while control follicles grew by a further 0.8 mm over the subsequent 6 days. In contrast, 10 nM TPA treatment did not affect hair fibre elongation for a period of 8 days, after which TPA-treated fibre production ceased while control fibres grew by a further 0.79 mm over the subsequent 7 days. Incubation of hair follicles with TPA resulted in a 41% inhibition of hair fibre protein synthesis, as measured biochemically from the incorporation of 3H-leucine using a differential akali extraction method. The inhibitory effect of TPA on follicle growth was partially prevented by preincubation with the selective PKC inhibitor H-7, and almost completely prevented by preincubation with the more potent PKC inhibitor Ro 31-7549. Neither agent alone significantly affected follicle growth at concentrations that reversed the TPA response. These findings indicate that PKC is a negative regulator of hair follicle growth, and suggest that PKC may play a part in the transduction of follicular growth-inhibitory signals.

Biphasic effect of 1,25-dihydroxyvitamin D3 on human hair follicle growth and hair fiber production in whole-organ cultures.

We have used a whole-organ culture system to investigate the effects of 1,25(OH)2D3 on human hair follicle growth and hair fiber production. Relatively low concentrations (1-10 nM) of 1,25(OH)2D3 stimulated the cumulative growth of hair follicles and hair fibers, by 52% and 36%, respectively (concentration producing 50% of the maximal response [EC50] values of 0.3 nM). The initial rates of follicle and fiber growth were increased, whereas the respective growth periods were unaffected. At higher concentrations of 1,25(OH)2D3, there was a dose-dependent inhibition of both follicle and fiber growth (IC50 values of 100 nM), in part due to reduction in the growth periods. There was a marked delay between the onset of 1,25(OH)2D-induced hair follicle and hair fiber growth inhibition. Incubation of hair follicles with 100 nM 1,25(OH)2D3 resulted in a rapid, transient inhibition of DNA synthesis (55% inhibition at 24 h), followed by a gradual return to control levels at day 4. Prolonged (> 5 h), incubation in the presence of 100 nM of 1,25 (OH)2D3 was required for follicle growth inhibition to be manifest. Ro 31-7549, a selective inhibitor of protein kinase C, did not prevent 1,25(OH)2D3-induced inhibition of hair follicle growth. These data suggest that 1,25(OH)2D3 may play a physiologic role in maintaining optimal hair follicle activity, and that elevation of 1,25(OH)2D3 may inhibit hair growth in vivo.

Hair fibre production by human hair follicles in whole-organ culture.

We have used both pulse-chase radiolabelling and morphometric techniques to investigate the ability of whole-organ cultures of human hair follicles to synthesize hair fibre. Anagen hair follicles were obtained from human facial skin by microdissection. Follicles were pulse-labelled with 3H-leucine, and radioactivity in alkali-soluble protein (ASP) and alkali-insoluble hair fibre protein (HFP) fractions was determined by differential extraction. There was a slow decline in ASP radioactivity over 7 days of culture, with a half-life of 4 days. In contrast, leucine incorporation into HFP increased linearly from day 1 to day 5, following an initial delay of 17 h. Morphometric analysis revealed that hair follicles and hair fibres grew at the same rate (0.25 mm/day) for 4 days in culture, after which the rate of hair follicle growth declined progressively, whereas fibre growth continued at the initial rate for a further 3 days. Concurrent with the onset of the decline in follicle growth, the base of the hair fibre began to descend towards the follicle base, until hair fibre occupied almost the full length of the hair follicle at day 15. The decline in follicle growth was preceded by a decline in the rate of follicular 3H-thymidine incorporation, with a delay of approximately 1 day. Overall, these data demonstrate that human hair follicles in whole-organ culture are capable of the production of hair fibre, and suggest that cessation of hair growth in vitro results from depletion of the pool of differentiated follicular keratinocytes, secondary to a loss of matrix cell proliferative activity.

IL-1 alpha inhibits human hair follicle growth and hair fiber production in whole-organ cultures.

In this study we have used a recently developed human hair follicle whole-organ culture system to investigate the effect of IL-1 alpha on hair follicle growth and hair fiber production. In the presence of 10 ng/ml IL-1 alpha, the growth of cultured human hair follicles ceased within 2-4 days, whereas control hair follicles grew for a period of 7-10 days. IL-1 alpha also inhibited hair fiber growth, but with an onset which occurred 3 days later than that of follicle growth inhibition. An IC50 value of approximately 30 pg/ml was obtained for IL-1 alpha inhibition of follicle growth. Incubation of hair follicles with IL-1 alpha resulted in a rapid, transient reduction in the rate of whole-follicle DNA synthesis. 1000-fold molar excess of IL-1 receptor antagonist prevented IL-1-induced follicle growth inhibition, while antagonist alone was without effect. The selective PKC inhibitor, Ro 31-7549, augmented IL-1-induced inhibition of hair follicle growth, but did not itself affect hair follicle growth. These findings indicate that IL-1 alpha exerts a rapid antiproliferative effect on hair follicles, and that inhibition of hair fiber growth is a secondary response. Thus, IL-1 may play a role in the pathophysiology of inflammatory hair loss conditions, such as alopecia areata, through a direct growth-inhibitory effect on hair follicles.