Abstract
Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1β mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and ZIPK have diverse, but predominantly distinct regulatory functions in vascular SMC and that ROCK1-mediated activation of ZIPK is not involved in most of these functions.
Highlights
Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) are serine/ threonine protein kinases that have been implicated in a variety of important physiological functions, including smooth muscle contraction, cell proliferation, cell adhesion, apoptosis, cell migration and inflammation [1,2,3,4,5,6].ROCK belongs to a kinase family that is primarily activated by interaction with the small GTPase RhoA [7,8,9]
coronary artery smooth muscle cells (CASMC) and umbilical artery smooth muscle cells (UASMC) were co-transfected with FITClabeled control siRNA and either ROCK1- or ZIPK-targeted or control siRNA, nuclei were labeled with DAPI and transfected cells were detected by fluorescence confocal microscopy
We examined the effect of ROCK1 knockdown on ROCK2 expression and found that ROCK1 knockdown decreased ROCK2 protein expression to 65.2 ± 9.1% of control (n = 6)
Summary
Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) are serine/ threonine protein kinases that have been implicated in a variety of important physiological functions, including smooth muscle contraction, cell proliferation, cell adhesion, apoptosis, cell migration and inflammation [1,2,3,4,5,6].ROCK belongs to a kinase family that is primarily activated by interaction with the small GTPase RhoA [7,8,9]. In contrast to the other DAPKs, ZIPK does not contain a calmodulin-binding domain, consistent with the fact that its activity is not regulated by Ca2+ [13] It lacks a death domain found in other family members, but does contain three nuclear localization signals. Several substrates of ZIPK have been identified, including STAT3 (signal transducer and activator of transcription 3) [17], myosin II regulatory light chains (LC20) [18,19], CPI-17 (the 17-kDa protein kinase C-potentiated inhibitory protein of type 1 protein serine/threonine phosphatase) [20] and MYPT1 [18,21]. DAPK1 [23] and ROCK1 [24] have been implicated as ZIPK kinases and Thr265 has been identified as a ZIPK autophosphorylation site whose phosphorylation is required for full catalytic activity and cell death [25]. ZIPK expression is increased in the aorta and mesenteric artery of spontaneously hypertensive compared to normotensive Wistar Kyoto rats [26] and ZIPK has been shown to mediate reactive oxygen species (ROS)-dependent vascular inflammation, potentially leading to hypercontractility and hypertrophy associated with hypertension [27]
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