Transient activation of beta-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

When beta-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for beta-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised beta-catenin to the ligand-binding domain of a mutant oestrogen receptor (DeltaNbeta-cateninER). DeltaNbeta-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and beta-catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of beta-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous beta-catenin signalling is required to maintain hair follicle tumours.     

The hair cycle and Vitamin D receptor

The Vitamin D receptor (VDR) plays a critical role in epidermal homeostasis. The ligand-dependent actions of the VDR attenuate epidermal keratinocyte proliferation and promote keratinocyte differentiation. Calcium can compensate for the absence of the VDR in maintaining a normal program of epidermal keratinocyte differentiation both in vitro and in vivo. In contrast, the effects of VDR ablation on the hair follicle cannot be prevented by maintaining normal calcium levels and are independent of 1,25-dihydroxyvitamin D. These actions of the VDR are critical in the keratinocyte stem cell population that resides in the bulge region of the hair follicle. Absence of a functional VDR leads to a self-renewal and lineage progression defect in this population of stem cells, resulting in the absence of post-morphogenic hair cycles. The molecular partners and downstream target genes of the VDR in this unique population of cells have not yet been identified.     

Smad7-induced beta-catenin degradation alters epidermal appendage development

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.     

Role of the vitamin D receptor in hair follicle biology

The vitamin D receptor (VDR) is expressed in numerous cells and tissues, including the skin. The critical requirement for cutaneous expression of the VDR has been proven by investigations in mice and humans lacking functional receptors. These studies demonstrate that absence of the VDR leads to the development of alopecia. The hair follicle is formed by reciprocal interactions between an epidermal placode, which gives rise to the hair follicle keratinocytes and the underlying mesoderm which gives rise to the dermal papilla. Hair follicle morphogenesis ends the second week of life in mice. Studies in VDR null mice have failed to demonstrate a cutaneous abnormality during this period of hair follicle morphogenesis. However, VDR null mice are unable to initiate a new hair cycle after the period of morphogenesis is complete, therefore, do not grow new hair. Investigations in transgenic mice have demonstrated that restricted expression of the VDR to keratinocytes is capable of preventing alopecia in the VDR null mice, thus demonstrating that the epidermal component of the hair follicle requires VDR expression to maintain normal hair follicle homeostasis. Studies were then performed to determine which regions of the VDR were required for these actions. Investigations in mice lacking the first zinc finger of the VDR have demonstrated that they express a truncated receptor containing an intact ligand binding and AF2 domain. These mice are a phenocopy of mice lacking the VDR, thus demonstrate the critical requirement of the DNA binding domain for hair follicle homeostasis. Transgenic mice expressing VDRs with mutations in either the ligand-binding domain or the AF2 domain were generated. These investigations demonstrated that mutant VDRs incapable of ligand-dependent transactivation were able to prevent alopecia. Investigations are currently underway to define the mechanism by which the unliganded VDR maintains hair follicle homeostasis.     

Autocrine TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in breast cells

In this study, we address whether TGFbeta signaling mediates vitamin D3 analog-induced growth inhibition in nonmalignant and malignant breast cells. Normal mammary epithelial cells (184), immortalized nonmalignant mammary epithelial cells (184A1 and MCF10A), and breast cancer cells (early passage MCF7: MCF7E) were sensitive to the inhibitory effects of vitamin D3 analogs (EB1089 and MC1288) while late passage MCF7 breast cancer (MCF7L) cells were relatively resistant. A similar pattern of sensitivity to TGFbeta was observed with these cells. Thus, the sensitivity to the vitamin D3 analogs correlated with the sensitivity to TGFbeta. MCF7L TGFbetaRII-transfected cells, which have autocrine TGFbeta activity, were more sensitive to EB1089 than MCF7L cells. TGFbeta neutralizing antibody was found to block the inhibitory effects of these analogs. These results are consistent with the idea that autocrine TGFbeta signaling mediates the anti-proliferative effects of the vitamin D3 analogs in these cells. The expression of TGFbeta isoforms and/or TGFbeta receptors was induced by the analogs in the vitamin D3 and TGFbeta sensitive cells. Vitamin D3 analogs did not induce TGFbeta or TGFbeta receptor expression in the resistant MCF7L cells. Therefore, EB1089 induces autocrine TGFbeta activity through increasing expression of TGFbeta isoforms and/or TGFbeta receptors. In addition, EB1089 induced nuclear VDR protein levels in the sensitive 184A1 cells but not in the resistant MCF7L cells. 184A1 cells were more sensitive to EB1089-induced VDR-dependent transactivation than MCF7L cells as measured by a luciferase reporter construct containing the VDRE, indicating a defect of VDR signaling in MCF7L cells. Smad3, a TGFbeta signaling mediator, coactivated VDR-dependent transactivation in 184A1 cells but not in MCF7L cells. These results indicate that Smad3 coactivates VDR to further enhance TGFbeta signaling and vitamin D3 signaling in the sensitive 184A1 cells. The results also indicate that Smad3 is not of itself sufficient to coactivate VDR in TGFbeta/vitamin D3 resistant MCF7L cells and other factors are required. We found that the PI 3-kinase pathway inhibitor LY29004 inhibited the synergy of TGFbeta and EB1089 on VDR-dependent transactivation activity. This indicates that the crosstalk between TGFbeta and vitamin D signaling is also PI 3-kinase pathway dependent.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog,EB 1089

The standard of care for unresectable lung cancer is chemoradiation. However, therapeutic options are limited and patients are rarely cured. We have previously shown that vitamin D and vitamin D analogs such as EB 1089 can enhance the response to radiation in breast cancer through the promotion of a cytotoxic form of autophagy. In A549 and H460 non-small cell lung cancer (NSCLC) cells, 1,25-D₃ (the hormonally active form of vitamin D) and EB 1089 prolonged the growth arrest induced by radiation alone and suppressed proliferative recovery, which translated to a significant reduction in clonogenic survival. In H838 or H358 NSCLC cells, which lack VDR/vitamin D receptor or functional TP53, respectively, 1,25-D₃ failed to modify the extent of radiation-induced growth arrest or suppress proliferative recovery post-irradiation. Sensitization to radiation in H1299 NSCLC cells was evident only when TP53 was induced in otherwise tp53-null H1299 NSCLC cells. Sensitization was not associated with increased DNA damage, decreased DNA repair or an increase in apoptosis, necrosis, or senescence. Instead sensitization appeared to be a consequence of the conversion of the cytoprotective autophagy induced by radiation alone to a novel cytostatic form of autophagy by the combination of 1,25-D₃ or EB 1089 with radiation. While both pharmacological and genetic suppression of autophagy or inhibition of AMPK phosphorylation sensitized the NSCLC cells to radiation alone, inhibition of the cytostatic autophagy induced by the combination treatment reversed sensitization. Evidence for selectivity was provided by lack of radiosensitization in normal human bronchial cells and cardiomyocytes. Taken together, these studies have identified a unique cytostatic function of autophagy that appears to be mediated by VDR, TP53, and possibly AMPK in the promotion of an enhanced response to radiation by 1,25-D₃ and EB 1089 in NSCLC.     

Dermal β-catenin activity in response to epidermal Wnt ligands is required for fibroblast proliferation and hair follicle initiation

Dermal fibroblasts are required for structural integrity of the skin and for hair follicle development. Uniform Wnt signaling activity is present in dermal fibroblast precursors preceding hair follicle initiation, but the functional requirement of dermal Wnt signaling at early stages of skin differentiation and patterning remains largely uncharacterized. We show in mice that epidermal Wnt ligands are required for uniform dermal Wnt signaling/β-catenin activity and regulate fibroblast cell proliferation and initiation of hair follicle placodes. In the absence of dermal Wnt signaling/β-catenin activity, patterned upregulation of epidermal β-catenin activity and Edar expression are absent. Conversely, forced activation of β-catenin signaling leads to the formation of thickened dermis, enlarged epidermal placodes and dermal condensates that result in prematurely differentiated enlarged hair follicles. These data reveal functional roles for dermal Wnt signaling/β-catenin in fibroblast proliferation and in the epidermal hair follicle initiation program.     

Expression profiling in squamous carcinoma cells reveals pleiotropic effects of vitamin D3 analog EB1089 signaling on cell proliferation,differentiation, and immune system regulation

The active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is key mediator of calcium homeostasis and is a component of the complex homeostatic system of the skin. 1,25-(OH)2D3 regulates cellular differentiation and proliferation and has broad potential as an anticancer agent. Oligonucleotide microarrays were used to assess profiles of target gene regulation at several points over a 48 h period by the low calcemic 1,25-(OH)2D3 analog EB1089 in human SCC25 head and neck squamous carcinoma cells. One hundred fifty-two targets were identified, composed of 89 up- and 63 down-regulated genes distributed in multiple profiles of regulation. Results are consistent with EB1089 driving SCC25 cells toward a less malignant phenotype, similar to that of basal keratinocytes. Targets identified control inter- and intra-cellular signaling, G protein-coupled receptor function, intracellular redox balance, cell adhesion, and extracellular matrix composition, cell cycle progression, steroid metabolism, and more than 20 genes modulating immune system function. The data indicate that EB1089 performs three key functions of a cancer chemoprevention agent; it is antiproliferative, it induces cellular differentiation, and has potential genoprotective effects. While no evidence was found for gene-specific differences in efficacy of 1,25-(OH)2D3 and EB1089, gene regulation by 1,25-(OH)2D3 was generally more transient. Treatment of cells with 1,25-(OH)2D3 and the cytochrome P450 inhibitor ketoconazole produced profiles of regulation essentially identical to those observed with EB1089 alone, indicating that the more sustained regulation by EB1089 was due to its resistance to inactivation by induced 24-hydroxylase activity. This suggests that differences in action of the two compounds arise more from their relative sensitivities to metabolism than from differing effects on VDR function.     

A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR). Here we describe a patient with HVDRR who also exhibited some hypotrichosis of the scalp but otherwise had normal hair and skin. A 102 bp insertion/duplication was found in the VDR gene that introduced a premature stop (Y401X). The patient's fibroblasts expressed the truncated VDR, but were resistant to 1,25(OH)2D3. The truncated VDR weakly bound [3H]-1,25(OH)2D3 but was able to heterodimerize with RXR, bind to DNA and interact with the corepressor hairless (HR). However, the truncated VDR failed to bind coactivators and was transactivation defective. Since the patient did not have alopecia or papular lesions of the skin generally found in patients with premature stop mutations this suggests that this distally truncated VDR can still regulate the hair cycle and epidermal differentiation possibly through its interactions with RXR and HR to suppress gene transactivation.