Date Published: December 4, 2009
Publisher: Public Library of Science
Author(s): Frances E. Lock, Neil A. Hotchin, Joy Sturtevant. http://doi.org/10.1371/journal.pone.0008190
Abstract: The human epidermis is comprised of several layers of specialized epithelial cells called keratinocytes. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated. The mammalian serine/threonine kinases (ROCK1 and ROCK2) are well-characterised downstream effectors of the small GTPase RhoA. We have previously demonstrated that the RhoA/ROCK signalling pathway plays an important role in regulation of human keratinocyte proliferation and terminal differentiation. In this paper we addressed the question of which ROCK isoform was involved in regulation of keratinocyte differentiation.
Partial Text: The human epidermis is comprised of several layers of specialized epithelial cells called keratinocytes. As keratinocytes are lost from the outermost epidermal layers, they are replaced through a process of terminal differentiation in which keratinocytes in the basal layer exit the cell cycle, down-regulate adhesion to the extracellular matrix (ECM) proteins of the basal lamina and migrate upwards through the supra-basal, differentiated layers, until they eventually reach the outermost cornified layer . The basal lamina is made up of various ECM proteins, including fibronectin, collagens and laminins. Keratinocytes in the basal layer of the epidermis adhere to these ECM proteins via integrin adhesion receptors and there is considerable evidence that adhesion to ECM plays a key role in regulating epidermal function . Disruption of integrin-ECM interactions results in initiation of keratinocyte terminal differentiation in vitro –. Hence, normal epidermal function requires that the balance between keratinocyte proliferation, adhesion to ECM proteins and terminal differentiation be tightly regulated. Previous data from our laboratory and others suggest that signalling though Rho family GTPases is required for keratinocyte terminal differentiation –. RhoA is a member of the Rho family of small GTPases and acts as a molecular switch to regulate a plethora of cellular processes including organisation of the actin cytoskeleton, cell adhesion and motility and gene expression . The best-characterised downstream effectors of RhoA are the serine/threonine kinases ROCK1 and ROCK2 (also known as ROKβ and ROKα, respectively) , . Both ROCK isoforms are comprised of an N-terminal region, a kinase domain, a coiled-coil domain containing a Rho binding site, a PH domain and a C-terminal domain . Both isoforms share a high amino acid sequence identity, with 92% identity across their kinase domains. However, the two kinases only share 65–70% sequence identity across their PH domains, which may account for the observed differences in cellular localisation of the two isoforms , , . Most studies to date have either used over-expression of ROCK or pharmacological inhibition of ROCK , , . Neither of these methods allows discrimination of isoform-specific functions. Recently, functional differences between the two ROCK isoforms have become more apparent. In vivo data show that, despite their structural similarities, ROCK1 or ROCK2 expression cannot compensate for loss of the other isoform during murine embryonic development –. In vitro studies utilising ROCK isoform specific RNAi knockdown in fibroblasts also suggest that ROCK1 and ROCK2 may have distinct, and sometimes opposing, roles in the cell , . In this study we used RNAi to specifically knockdown ROCK1 or ROCK2 expression in cultured keratinocytes and analysed adhesion to various ECM proteins and the differentiation status of the cells. Our data suggest that both ROCK isoforms play distinct and important roles in regulating keratinocyte differentiation status and keratinocyte adhesion to the ECM protein fibronectin.
HaCaT keratinocytes were stably transfected with GFP-IRES-shRNAmir constructs specifically targeting ROCK1 or ROCK2 or a non-silencing control nonsense mRNA sequence (NSC) to generate HaCaT-ROCK1-KD, HaCaT-ROCK2-KD and HaCaT-NSC cells respectively. A stable decrease in ROCK1 expression was observed in HaCaT-ROCK1-KD cells, compared to HaCaT-NSC and HaCaT-ROCK2-KD cells (Figure 1A, B). Similarly, a significant decrease in ROCK2 expression was observed in HaCaT-ROCK2-KD cells, when compared to HaCaT-NSC and HaCaT-ROCK1-KD cells (Figure 1C, D). Depletion of ROCK1 or ROCK2 had no effect on expression of the other, non-targeted, ROCK isoform (Figure 1A, C). To further characterise these cell lines following ROCK isoform knockdown, HaCaT-NSC, HaCaT-ROCK1-KD and HaCaT-ROCK2-KD cell lysates were immunoblotted to assess changes in phosphorylation of two known ROCK targets – myosin phosphatase (MYPT) and myosin light chain (MLC). Both ROCK1 and ROCK2 are able to directly phosphorylate MYPT1 on threonine residue 696 whereas serine 19 of MLC is phosphorylated by ROCK1 but not ROCK2 –. Decreased MYPT phosphorylation was observed in both HaCaT-ROCK1-KD and HaCaT-ROCK2-KD cells when compared to HaCaT-NSC cells (Figure 1E). Consistent with MLCpSer19 being a ROCK1 substrate but not a ROCK2 substrate, a decrease in phosphorylated MLC was observed in HaCaT-ROCK1-KD cells, but not in HaCaT-ROCK2-KD or HaCaT-NSC cells (Figure 1E). These results confirm that stable knockdown of ROCK1 and ROCK2 expression in these HaCaT keratinocyte cell lines is isoform specific and has functional consequences in terms of phosphorylation of known downstream effectors.
Our data confirm that both ROCK isoforms are expressed in cultured human keratinocytes and that each isoform can be specifically depleted, with no effect on the expression of the other (Figure 1A–D). We observed that depletion of either ROCK1 or ROCK2 results in decreased phosphorylation of MYPT on Thr696 (Fig 1E). This is consistent with published data where both ROCK1 and ROCK2 have been reported to phosphorylate MYPT on Thr696, leading to its inactivation –. In contrast, we observed a specific loss in phosphorylation of MLC on Ser19 in ROCK1 depleted cells but not in ROCK2 depleted cells (Figure 1E). Again, this is consistent with recent studies describing MLC as a ROCK1-specific target , , . This implies that continued expression of one isoform cannot compensate for the loss of the other, suggesting specific functional differences in human keratinocytes. Previous work from our laboratory has shown that the Rho/ROCK signalling pathway is important in regulating keratinocyte function . Here we have shown that the two ROCK isoforms have distinct roles in the regulation of keratinocyte adhesion to fibronectin (Figure 2A). One possible explanation for the differences in adhesion to fibronectin in ROCK1-depleted keratinocytes might be a consequence of loss of actinomyosin contractility affecting adhesion complexes. However, under the conditions used in the adhesion assays (1 hour adhesion) we observed no differences in adhesion complex size (data not shown). The role of ROCK1 function in adhesion to fibronectin has been analysed in rat embryo fibroblasts which, when seeded on fibronectin, displayed significantly higher ROCK1 activity than ROCK2 . This led the authors to conclude that the adhesion process has a particular requirement for ROCK1 . This would appear to be consistent with our data in which we see a decrease in adhesion to fibronectin in ROCK1-depleted keratinocytes but it is worth noting that adhesion to fibronectin-coated beads was unaffected in ROCK1-depleted fibroblasts . Alternatively, it might be a consequence of altered fibronectin matrix assembly, as has been observed in fibroblasts . However, this is unlikely to have been a factor in the relatively short period of adhesion used in our assays. It is unclear why adhesion to fibronectin is affected by depletion of ROCK1 or ROCK2 but adhesion to laminin-332 or collagen IV, both of which are known keratinocyte ECM ligands, is unaffected (Figure 2B,C). One possibility is that ROCK1 and ROCK2 regulate expression and/or function of keratinocyte fibronectin receptors (e.g. α5β1 integrin). We have analysed expression and function of the most abundant fibronectin-binding integrin, α5β1, but did not observe any consistent or significant changes in expression or activity (data not shown). This does not rule out the possibility that other fibronectin-binding integrins (e.g. αvβ5) are involved and this is currently being investigated.