Human TSC2-null fibroblast-like cells induce hair follicle neogenesis and hamartoma m

[IDW2BB]

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108033/

Our results indicate that TSC2-null fibroblast-like cells are the inciting cells for TSC skin hamartomas. When these cells are grafted onto mice as composites overlaid with normal newborn keratinocytes, the grafted skin manifests features of TSC skin tumours. TSC2-null cells directly or indirectly regulate multiple cell types, stimulating angiogenesis, recruiting mononuclear phagocytes, increasing epidermal proliferation, and inducing follicular neogenesis. The capacity of TSC2-null cells to induce normal keratinocytes to form follicular epithelium suggests that TSC hamartomas reflect ongoing attempts at foetal tissue morphogenesis, whereby tumour cells continue to convey inductive signals to other cells forming the tissue, as postulated by Sylvan Moolten in 194238.

TSC2-null cells from certain samples appeared to have a greater capacity for inducing hair follicles than others. Sources of variability include patient age (samples from patients under age 32 years induced follicles, whereas those from patients over 37 years did not) and tumour type (cells from a periungual fibroma, a tumour type that does not contain hair follicles, did not induce follicles). Cell passage number is also expected to influence results since hair follicle induction in using mouse dermal cells decreased with repeated passage of dermal cells39 or keratinocytes12. We grafted early passage cells, but it is notable that tumour cells that induced follicles were combined with passage 3 keratinocytes and those that failed to induce hair follicles were combined with passage 4-5 keratinocytes. Other potentially confounding factors are differences in mutations or developmental timing of second-hit mutations. Additional studies are required to determine the relative importance of these variables on follicle-inducing capacity.

TSC2-null cells overexpress certain genes that are characteristic of dermal papilla cells, so follicle-inducing tumour cells may share origins with hair follicle dermal cells. Some genes characteristically expressed by follicle-inducing cells, such as nestin, versican, and alkaline phosphatase, were not overexpressed by TSC2-null cells in vitro, but were clearly “turned on†in vivo in the follicular microenvironment. Differences in gene expression patterns between TSC2-null cells and murine dermal papilla cells might reflect differences between species or alterations in gene expression related to loss of TSC2 function. Further studies are necessary to determine the relationship between loss of TSC2 function and the capacity of these cells to induce hair-follicles, although studies of other cell lineages in mice suggest that loss of Tsc1/Tsc2 function alters differentiation of multipotent progenitor cells. The loss of Tsc2 in radial glia increases a progenitor pool and decreases numbers of neurons40, whereas deletion of Tsc1 in hematopoietic stem cells increases granulocyte-monocyte progenitors and decreases megakaryocyte-erythrocyte progenitors41. Loss of TSC2 function in skin tumour cells may skew differentiation of dermal cells towards follicle-inducing cells and/or promote the preservation of follicle-inducing capability in vitro.

Rapamycin decreased numbers of TSC2-null cells and mononuclear phagocytes, as well as angiogenesis and epidermal proliferation. These results suggest that the decreased redness and size of TSC skin lesions observed in patients receiving systemic21 or topical24 rapamycin may result from both anti-tumour cell effects and anti-angiogenic effects. The antiangiogenic effects of rapamycin may be due to direct inhibition of vascular endothelium and/or indirect effects such as diminished release of angiogenic factors by TSC2-null cells or recruitment of pro-angiogenic mononuclear cells. Rapamycin did not significantly affect hair follicle number or density. The lack of effect on hair follicle parameters may indicate that induction of follicles is mTORC1-independent, or that rapamycin was ineffective after follicular neogenesis had commenced.

TSC2-null cells from angiofibromas and fibrous plaques are tools for exploring follicular morphogenesis and regeneration. The fact that TSC skin tumours usually arise postnatally suggests the possibility of creating or amplifying the numbers of follicle-inducing cells via agents or stimuli impacting the TSC1/TSC2/mTORC1 pathway and/or signalling pathways involved in the genesis of other follicular hamartomas. Just as studies of cancers have revealed mechanisms of cellular growth and proliferation, studies of hamartomas should provide insights into tissue organization and maturation.

Free article.

Cotsarelis is working with Thangapazham.

- - - Updated - - -

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828374/

Clinically, patterned hair loss has been reported in non-Androgenetic Alopecia individuals with iatrogenic androgen stimulation [30], and in patients suffering from diseases characterized by excessive androgen, such as polycystic ovary syndrome and androgen-secreting tumors [31]. It is known that balding DPCs already showed varying degrees of premature senescence in early passages [18]. Moreover, DPCs isolated from non-balding areas of normal individuals have been demonstrated to be androgen-responsive and have been used as a cell model of Androgenetic Alopecia [32]. AR expression level is a crucial factor in determining DPC sensitivity to androgens in Androgenetic Alopecia [33]. It is known that AR mRNA [34], [35] and protein [36] are expressed in DPCs. In addition, DPCs isolated from balding area express more AR compared to those in non-balding areas [6], [37]. AR expression is stronger in early-passage DPCs and gradually decreasing during subcultivation [28]. Our results showed that earlier-passage DPCs with higher AR expression were more sensitive to androgen-mediated premature senescence. It is well known that primary DPCs spontaneously undergo replicative senescence during subcultivation. Indeed, we also observed that late-passage DPCs were more senescent (Fig. 2). Loss of responses to androgen-induced senescence in late-passage DPCs could reflect relatively lower AR expression in these cells as well as masking of androgen effects by replicative senescence. In our study, we also demonstrated that androgen-accelerated premature senescence was affected by AR expression level (Fig. 3 and ​and4).4). We thus concluded that blocking androgen/AR actions could play an important role in suppressing premature senescence in DPCs.

In prostate cancer cell lines, androgen signaling has been reported to induce recruitment of the AR-topoisomerase II beta (TOP2B) complex, which catalyzes DSBs at regulatory regions of AR target genes [20]. AR also acts in concert with genotoxic stress to induce alterations in local chromatin architecture. These events are permissive for sensitizing these regions to undergo chromosomal translocation through activation of ORF2 endonuclease [38]. In our study, DSBs were also induced in response to androgen/AR signaling, and γ-H2AX foci and expression levels of γ-H2AX proteins were further increased with AR overexpression (Figure 5). These results support previous findings that two important DNA damage sensors involved in the phosphorylation of H2AX–the active form of ATM (ataxia-telangiectasia-mutated kinase) and ATR (ATM and Rad3-related)–were detected only in balding DPCs [18]. Although much of this DNA damage can be repaired and the cell can then re-enter the cell cycle, some of the aberrantly enhanced DSBs might destabilize the genome and potentially trigger premature senescence in DPCs. The roles of TOP2B and ORF2 in DNA damage leading to premature senescence of DPCs need further investigation.

The effects of androgen/AR signaling on senescence in prostate cancer cells remain a matter of controversy [39], [40]. It has been reported that androgen depletion induces senescence in prostate cancer cells via down-regulation of Skp2 [39], [40]. In contrast, androgen/AR signaling has also been reported to drive cellular senescence without the involvement of DNA damage and p16INK4a upregulation in both prostate cancer and normal immortal prostate epithelial cell lines [39]. In DPCs, we found that p16INK4a protein levels were upregulated in response to androgen, and AR overexpression may further enhance the expression level of p16INK4a (Figure 3E). These results suggest that androgen/AR signaling promotes senescence through the p16INK4a pathway in DPCs and are in agreement with the results of a previous study, which showed increased expression of p16INK4a in balding DPCs from Androgenetic Alopecia patients with premature senescence [18]. The discrepant reports of androgen/AR actions in senescence could reflect the diversity of biological responses to androgen/AR signaling in different cell types. While these latter studies utilized the prostate cancer cell lines, PC3 and PC3-AR, and immortalized normal prostate RWPE-1 cells as experimental models, it is well known that PC3 cells are AR-, p16INK4a- and p53-null [39], and RWPE-1 cells are p53- and Rb-null [41]. Therefore, the senescence response and the pathway that mediates it in these cells might be different from that in primary DPCs. The signaling pathways activated by DNA damage converge on p53/p21 pathway-mediated replicative senescence caused by telomere shortening, and the p16 pathway is thought to mediate premature senescence [19]. Expression of p16INK4a is induced by numerous stressors, including oxidative stress [42], overexpression of oncogenes [43], [44], and DNA damage [45], [46]. Nuclear expression of oxidative stress and DNA damage markers has been reported in balding DPCs [18]. Increased DSBs and upregulation of p16INK4a in response to androgen/AR signaling suggest that DNA damage might be important in the androgen-accelerated premature senescence of DPCs, although we cannot exclude the possibility that oxidative stress is also induced by androgen/AR signaling. Androgen inducible molecules, such as Interleukin-6 (IL-6) and transforming growth factor (TGF)-β1 have been shown to inhibit hair growth in paracrine manner [27], [47] and could be contributing factors of cellular senescence. Whether the androgen/AR-accelerated premature senescence of DPCs is also mediated via autocrine manner by androgen inducible IL-6 and TGF-β1 needs more investigation.

The previous studies showed low passage DPCs could sustain epidermal cell proliferation; however, high passage DPCs could not [48]. In addition, DPCs after multiple passaging also reduced hair growth-promoting capabilities in vivo [30], [49]. These evidence supports the senescent DPCs may have functional defect on promoting epithelial-mesenchymal interactions. It has been shown that androgen/AR regulates the interaction of DPCs and follicular KCs by androgen-inducible factors secreted from DPCs [27], [28], [47], [50]. Recently, an impairment of hair follicle stem cells to differentiate into progenitor cells was reported to play a key role in the pathogenesis of Androgenetic Alopecia [51]. Miniaturization of hair follicles, the hallmark of Androgenetic Alopecia, displays thinner hair fibers and smaller DP size. It is possible that the androgen/AR-induced senescence in DPCs may not only lead to diminished DP size but also deregulate the communication between DPCs and hair follicle stem cells to differentiate to progenitor cells.

Here, we showed a previously unidentified relationship between androgen/AR signaling and induction of premature senescence in association with DNA damage and p16INK4a upregulation in DPCs. Our study highlights the importance of androgen/AR-accelerated premature senescence in DPCs, a process that is thought to reflect irreversible cell growth arrest in the progression of Androgenetic Alopecia. The acceleration of premature senescence of DPCs by androgen/AR signaling may explain the miniaturization of hair follicles shown in Androgenetic Alopecia patients. These results provide novel impacts of androgen/AR signaling in balding DPCs and offer the potential therapeutic targets on combating for Androgenetic Alopecia.