Distinct roles for ROCK1 and ROCK2 in the regulation of keratinocyte differentiation

Abstract

Background: 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.

Methodology and principal findings: We used RNAi to specifically knockdown ROCK1 or ROCK2 expression in cultured human keratinocytes. ROCK1 depletion results in decreased keratinocyte adhesion to fibronectin and an increase in terminal differentiation. Conversely, ROCK2 depletion results in increased keratinocyte adhesion to fibronectin and inhibits terminal differentiation.

Conclusion: These data suggest that ROCK1 and ROCK2 play distinct roles in regulating keratinocyte adhesion and terminal differentiation.

Figure 1. ROCK isoform-specific knockdown affects phosphorylation of downstream targets.

HaCaT-NSC, HaCaT-ROCK1-KD or HaCaT-ROCK2-KD cell lysates were immunoblotted to assess ROCK1 expression (A) or ROCK2 expression (C). B and D show densitometric analysis of knockdown of ROCK1 (B) or ROCK2 (D) relative to non-silencing (NSC) control cells and are the mean of 3 separate experiments (** p<0.01). Phosphorylation of myosin phosphatase-1 residue Thr696 (p-MYPT) and myosin light chain residue Ser19 (p-MLC) were also analysed and data shown are representative of 3 separate experiments (E).

Figure 2. ROCK isoform-specific knockdown regulates cell adhesion to fibronectin.

Adhesion of HaCaT-NSC, HaCaT-ROCK1-KD or HaCaT-ROCK2-KD to the extracellular matrix ligands fibronectin (A) collagen IV (B) and laminin 332 (C) was analysed. The mean and standard error of 3 separate experiments are shown in each case. Statistical analysis was carried out using unpaired two-way Student's T-test (** p<0.01).

Figure 3. ROCK isoform-specific knockdown regulates keratinocyte differentiation.

A, HaCaT-NSC, HaCaT-ROCK1-KD or HaCaT-ROCK2-KD cells were cultured for 2 days post-confluence and lysed. Expression of keratin 5, keratin 10 and tubulin were assessed by immunoblotting (A). SCC12f keratinocytes were transiently transfected with siRNA oligos to specifically knockdown ROCK1 or ROCK2 As a control SCC12f cells were transfected with a non-silencing control oligo (NSC). Cells were lysed and immunoblotted to assess ROCK isoform knockdown (B) or fixed and immunostained to assess involucrin expression (C). The means and standard errors from 3 separate experiments are shown. Statistical analysis was carried out using unpaired two-way Student's T-test, ** p<0.01, * p<0.05.