The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional.
BACKGROUND
We previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research.


METHODS
In this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression, proliferation, behaviour and lifespan in the presence and absence of Y-27632.


RESULTS
We demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely, keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification.


CONCLUSIONS
ROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research, as well as for regenerative and personalized medicine.
Sandra
S
Chapman
David H
DH
McDermott
Kui
K
Shen
Moon Kyoo
MK
Jang
Alison A
AA
McBride
D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D011725:Pyridines / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000577:Amides; D004791:Enzyme Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases