| Follicle stem cells residing in the bulge of hair follicles continuously self-renew and are necessary for the maintenance of skin homeostasis and for hair regeneration, follicle stem cells derived cells can also be recruited to repair epidermis following injury. The asymmetric cell division is a defining characteristic of stem cells that enables them to simultaneously perpetuate themselves and generate differentiated progeny. Defects in regulation of stem cell self-renewal could result in tumorigenesis or decreased capacity for tissue repair. The molecular mechanism underlying the follicle stem cells maintenance remains poorly understood.Our previous study showed that keratinocyte specific Smad4 knockout mouse developed skin tumors, which provide suitable mouse model for exploring how Smad4 regulate the self-renewal of follicle stem cells and the relationship with the skin tumors. In the current study, we found that Smad4 was expressed in telogen follicle stem cells which suggested important role in regulation of self-renewal of stem cells. LacZ staining showed that the K5 induced Cre recombinase expressed in follicle stem cells and could mediated the deletion of Smad4 in it. Disruption of Smad4 stimulated the proliferation of keratinocytes, leading to hyperplasia of interfollicular epidermis, hair follicles and sebaceous glands. The Smad4 mutant mouse developed skin tumors before 8 months after birth.Does the hypertrophy and tumorigenesis in Smad4 mutant have relationship with the self-renewal of the follicle stem cells? Utilizing the slow cycling characteristic of stem cell, we generated BrdU label-retaining cells (LRCs): Ten-day-old mice were injected with BrdU for a total of four injections to label mitotic cells. After a chase about 2 months, body cells cycled quickly and diluted the BrdU labelling, however follicle stem cells have slow cell cycle and still contained the label. We checked the BrdU labeling after a 60-days chase and found that the BrdU positive cells concentrated in the bulge of the control mice, however in Smad4 mutant the LRC zones were expanded. Most of the BrdU positive cells were Ki67 negative in the control mice indicating that the follicle stem cells were normal silent, but increased numbers of LRCs were co-labeled with Ki67 in Smad4 mutant (11.2±5.0% vs. 2.9±2.5%) demonstrating the overactivated of stem cells. The activation of stem cells could dilute the label relatively quickly, after a 77 day chase, LRCs were barely detected in Smad4 mutants, however, they were still detected in the control bulge of hair follicles. These results confirmed the overactivated of the follicle stem cells in Smad4 mutants.Increased proliferation of follicle stem cells ultimately led to loss of keratin 15 (K15) and CD34 expression. FACS by CD34 andα6-integrin also showed the decreased stem cell number in P72 Smad4 mutant mice compared with the control (2.0±1.3% vs.13.2±5.5%), which indicated the overactivation of the follicle stem cells may cause the lose of them.Loss of follicle stem cells led to decreased proliferation in 12-month-old Smad4 mutant epidermis and the hypertrophy phenotypes withered with time. Follicle stem cells respond rapidly to epidermal wounding by generating short lived transient amplifying cells responsible for acute wound re-epithelization. There were more proliferating keratinocytes at the wound edge in 2-month-old Smad4 mutant compared with that of the control. However, 10-month-old Smad4 mutant keratinocytes at wound edge showed much less proliferative compared with that of 2 months, which also supported that Smad4 deletion cause the overactivation and then expension of follicle stem cells.Increased evidence showed close relationship between follicle stem cells and skin tumorigenesis. In our current study, we showed that Smad4 deletion resulted in overactivated follicle stem cells which bring about more transit amplifying cells. The proliferative cells induced skin tumorigenesis before 8 months after birth. The proliferation of follicle stem cells were stimulated at the expense of self-renewal, and with the exhausting of transient proliferation cells there were almost no skin tumor developed in Smad4 mutant after 8 months. Follicle stem cells are multipotent and could differentiate to epidermis, hair follicle and sebaceous gland, coincident with which, Smad4 mutant mice developed several kinds if skin tumors including squamous cell carcinoma, basal cell carcinoma, papilloma and adenoma sebaceum. We concluded that the skin tumorigenesis in Smad4 mutant mice were largely due to the overactivation of follicle stem cells.Increased nuclear localization of active-β-catenin was correlated with the overactivation of follicle stem cells, however the expression of Pten, p-Akt and p-GSK-3βwere not altered, which indicated that activation ofβ-catenin was independent of overactivation of PI3K/Akt signaling. Smad4 deletion also increased expression of c-Myc and decreasedα6-integrin expression, which might reduce adhesive interactions with the local microenvironment and alter the normal self-renewal of follicle stem cells. Normally, stem cell divide asymmetrically enables them to simultaneously perpetuate themselves and generate transit amplifying cells for differentiating, we proposed that Smad4 deletion drives stem cell divide into two transit amplifying cells at the expense of self-renewal and thereby stimulates proliferation.In conclusion, our data provided the first genetic evidence to show that Smad4 deletion caused overactivation of follicle stem cells and then resulted in skin tumorigenesis, demonstrating that Smad4 plays a pivotal role in follicle stem cell maintenance.
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