Human Keratinocyte Stem Cells Research Articles

Rapamycin (sirolimus) inhibits proliferating cell nuclear antigen expression and blocks cell cycle in the G1 phase in human keratinocyte stem cells.

Because the immunosuppressant rapamycin (sirolimus) blocks T cell proliferation in G1 phase, it has been proposed as a potential treatment for psoriasis, a skin disease characterized by T cell activation and keratinocyte stem cell hyperproliferation. To determine another potentially important mechanism through which rapamycin can act as an antipsoriatic agent, we tested its direct effect on keratinocyte stem cell proliferation in vitro as well as in vivo. In vivo cell cycle quiescent (G0 phase) stem cell keratinocytes in primary culture sequentially express de novo cyclin D1 and proliferating cell nuclear antigen (PCNA), prior to S phase entry, and upregulate beta1 integrin. Rapamycin inhibited the growth of keratinocytes that were leaving quiescence as well as those already in cell cycle without affecting cell viability. Although beta1 integrin(bright) expression was not affected, the number of beta1 integrin(bright) cells entering S/G2/M was significantly lowered by rapamycin. Cells treated with rapamycin exhibited decreased PCNA expression while cyclin D1 expression, which precedes PCNA expression in the cell cycle, was not affected. We found similar effects on stem cell keratinocytes in patients with psoriasis treated systemically with rapamycin. Because PCNA is required for cell cycle progression from G1 to S phase, our data indicate that inhibition of PCNA protein synthesis may be an important regulatory element in the ability of rapamycin to exert a G1 block.

Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes.