This report describes the effect of a ROCK inhibitor, Y-27632, on keratinocyte proliferation, immortalization, and differentiation. Y-27632 treatment was shown to result in the bypass of senescence and immortalization of different types of keratinocytes from human foreskin and vaginal and cervical epithelium. Preliminary experiments indicated that human foreskin fibroblasts treated with Y-27632 did not show enhanced proliferation or bypass senescence (see Supplemental Figure 7), suggesting that this is a keratinocyte specific phenomenon. These observations have far-reaching implications for the study and treatment of various skin diseases; improved culture and extended lifespan of keratinocytes will prove invaluable for both research and therapeutic purposes.
To date, our results show that Y-27632–immortalized cells are functionally equivalent to normal cells. They have a normal karyotype and an intact DNA damage response and are able to form a stratified epithelium in organotypic culture. The immortalized keratinocytes demonstrate upregulated telomerase mRNA levels and telomeres that have shortened but remain at a stable length. MYC protein levels increased with continued passage, and this may be responsible for upregulation of TERT
mRNA expression. Honma et al. have shown that Y-27632 rapidly induces MYC
mRNA expression in human keratinocytes (28
), and this might explain the initial induction of MYC that we observed in the first passage with Y-27632.
Previous studies have shown that telomerase expression is not sufficient for keratinocyte immortalization and inactivation of p16INK4A may also be required to bypass senescence in keratinocytes (7
). Y-27632–immortalized keratinocytes express low levels of the p16INK4A protein, but we assume that the cells are either resistant to its antiproliferative effect or protein levels are too low to mediate an inhibitory response. Rea et al. also observed p16INK4A expression in spontaneously immortalized keratinocytes cell lines (29
In this study, we have identified 2 factors to be important for keratinocyte immortalization: (a) culture with feeder fibroblasts and (b) exposure to a Rho kinase inhibitor. We have also observed that MYC is consistently upregulated during immortalization. These factors regulate the equilibrium between keratinocyte proliferation and differentiation. Prior studies have shown that culture of keratinocytes with feeder cells enhances lifespan, possibly in part by inducing telomerase (17
). However, culture of keratinocytes with feeder cells also results in the expression of differentiation markers in a subset of cells when compared with culture of these cells on plastic (26
). As shown in Supplemental Figure 7, Y-27632 reduced this tendency in monolayer culture. Moreover, Honma et al. propose that the Rho-GTPase pathway may control MYC activity (28
). MYC has a positive role in keratinocyte proliferation but also can promote differentiation of epidermal stem cells (30
). We postulate that the interaction of these pathways is important for Y-27632–mediated immortalization.
Future studies will address the mechanism of Y-27632–mediated immortalization of keratinocytes. ROCK can phosphorylate a number of different downstream targets and has profound effects on cell behavior. We do not yet know exactly which of these pathways are important for keratinocyte immortalization. However, despite the diverse effects of Y-27632, cells cultured with Y-27632 have many normal characteristics and are able to differentiate normally when Y-27632 is removed from the culture medium. Y-27632 can also inhibit other Rho effector kinases, such as citron kinase and PKN, but its affinity for ROCK kinase is 20- to 30-times higher (31
). Some of the observed effect of Y-27632 on keratinocytes could also be mediated by effects of ROCK inhibitor on the feeder fibroblasts, since efficient immortalization is only observed in their presence.
The current study shows many parallels between Y-27632–induced immortalization and HPV-mediated immortalization. In both cases, telomerase was induced and the cells efficiently bypassed senescence. Another parallel is the finding by Charette and McCance that both HPV16 E7 protein and Y-27632 treatment of human foreskin keratinocytes resulted in increased cell migration (32
). However, Y-27632–immortalized cells have a normal DNA damage response and karyotype compared with HPV-immortalized cells and are therefore preferable for many studies.
Keratinocytes are the natural host cells for all HPVs, but nononcogenic HPVs are difficult to study because they do not immortalize these cells. Thus, Y-27632–treated keratinocytes will be very useful for long-term analysis of nononcogenic HPV-related studies. Although not associated with cancer, these viruses are responsible for a great burden of recalcitrant disease, such as genital warts, respiratory papillomatosis, and cutaneous warts. These lesions can be especially problematic in individuals who are immunocompromised by HIV infection or organ transplantation. Y-27632–immortalized keratinocytes that can differentiate and support the entire viral life cycle will allow testing of antiviral therapies in a system that closely reflects the in vivo situation.
Human foreskin keratinocytes are most often used for HPV studies, because of the availability of this tissue from routine circumcision and difficulties in obtaining sufficient numbers of cells from other tissues. However, these keratinocytes might not be the best host for papillomavirus studies, as HPV infection of the foreskin is often clinically inapparent. A more appropriate cell type for the study of the many HPVs is that of the uterine cervix. We show here that Y-27632 can immortalize vaginal and cervical keratinocytes, making such cells much more amenable to study. Different HPVs have a very specific tropism for different regions of epithelia. Y-27632 treatment and expansion of small numbers of keratinocytes derived from different types of epithelia could greatly increase our understanding and treatment of HPV infection.
The ability to greatly expand and derive tissue from different types of primary human keratinocytes is of great research and therapeutic benefit. In an era in which personalized medicine is becoming increasingly important, patient-derived keratinocytes or tissue equivalents could be used to test therapies specific for the donor. Isolation and rapid expansion of keratinocytes from a small tissue biopsy using culture with Y-27632 could greatly increase the efficiency and time frame of this process. For example, small number of keratinocytes can be isolated, expanded in monolayer culture, and developed into tissue sheets for autologous epidermal replacement of regions with extensive burns or ulcers (33
). Keratinocyte-mediated gene therapy is another intensively studied area of research, whereby therapeutic genes can be introduced into autologous keratinocytes and regrafted onto the host (34
Transplantation of human keratinocytes, with enhanced in vitro proliferation, onto human hosts raises concerns of potential tumorigenic conversion. However, our studies have shown that ROCK inhibitor–treated cells have a normal p53-mediated growth arrest response and have no gross genetic abnormalities. Furthermore, there is evidence that ROCK inhibitors prevent rather than promote tumor progression and metastasis in human and animal models (35
). The Rho/ROCK pathway has been shown to function in the cardiovascular system, central nervous system, cancers, and embryonic development (37
). This pathway is an important therapeutic target, and a ROCK inhibitor (fasudil) is already marketed for cerebral vasospasm after surgery (38
) and is currently being tested for the treatment of angina pectoris, acute cerebral thrombosis, and other vascular diseases. Our study demonstrates that ROCK inhibitors may also be useful for quickly generating large numbers of normal keratinocytes and tissue equivalents that may be used in a variety of medical and research applications.