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. 2015 Feb 27;10(2):e0116969.
doi: 10.1371/journal.pone.0116969. eCollection 2015.

The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells

Jing-Ti Deng  1 , Xiu-Ling Wang  2 , Yong-Xiang Chen  3 , Edward R O'Brien  3 , Yu Gui  4 , Michael P Walsh  5
Affiliations

The effects of knockdown of rho-associated kinase 1 and zipper-interacting protein kinase on gene expression and function in cultured human arterial smooth muscle cells

Jing-Ti Deng et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Knockdown of ROCK1 and ZIPK at the protein level in CASMC.
CASMC were transfected with siRNAs to ROCK1 or ZIPK or with negative control siRNAs. Cells were lysed 48 h later for western blotting with anti-ROCK1 and anti-ZIPK. Loading levels were normalized using anti-GAPDH. Representative results are shown for two of a total of 10 independent experiments. Quantitative data are presented in Table 2.
Fig 2
Fig 2. Gene expression profiles.
Heat maps to indicate the most differentially expressed genes in (A) ROCK1 knockdown (> 2.4-fold change) and (B) ZIPK knockdown (> 2-fold change) CASMC. Colored bands represent the change of the indicated gene expression: down-regulation green and up-regulation red. The key to decipher the color is shown below the clustering image. Results from the triplicate analyses are included.
Fig 3
Fig 3. Effects of ROCK1 and ZIPK knockdown on IL-6 and IL-1β mRNA expression and IL-6 protein secretion.
(A) CASMCs were transfected with siRNA to ZIPK or ROCK1 or with negative control siRNA. Cells were lysed 48 h later for qRT-PCR to quantify IL-6 and IL-1β mRNA levels. Values represent means ± SEM (n = 7 for IL-6 and n = 6 for IL-1β). *significantly different from control (p < 0.001 except for IL-1β in ZIPK knockdown where p = 0.007). (B) IL-6 levels in the medium of control cells and cells transfected with ROCK1 or ZIPK siRNA were quantified by ELISA. Values represent means ± SEM (n = 20 for CASMC, n = 29 for UASMC). *significantly different from control (p < 0.002).
Fig 4
Fig 4. Effects of ROCK1 and ZIPK knockdown on IL-1β protein expression.
IL-1β protein expression in control cells and cells transfected with ZIPK or ROCK1 siRNA was examined by western blotting. (A) Two representative western blots showing the levels of IL-1β in control UASMC and in UASMC 48 h after transfection with siRNAs to ZIPK or ROCK1. Two loading controls were used: α-actin and SM-22. (B) Quantification of IL-1β expression levels relative to α-actin (upper panel) and SM-22 (lower panel) in control, ZIPK- and ROCK1-knockdown UASMC. Values represent means ± SEM (n = 13 for α-actin and n = 9 for SM-22). *significantly different from control (p < 0.05). In the case of ZIPK knockdown, statistical significance was not achieved (p = 0.54 for α-actin and p = 0.15 for SM-22).
Fig 5
Fig 5. Effects of ROCK1 and ZIPK knockdown on vascular smooth muscle cell viability.
The MTT cell viability assay was performed on CASMC and UASMC 48 h after transfection with siRNA to ROCK1 or ZIPK or with negative control siRNA. Values represent the means ± SEM (n = 8). *significantly different from control (p < 0.001).
Fig 6
Fig 6. Effects of ROCK1 and ZIPK knockdown on cell proliferation.
BrDU incorporation was observed by anti-BrDU staining in UASMC 48 h following transfection with siRNA to ZIPK or ROCK1 or with negative control siRNA. (A) Representative fields of cells are shown stained with anti-BrDU (top panels) or Hoechst nuclear stain (middle panels) with merged images shown in the bottom panels. (B) Quantitative data showing the numbers of BrDU-positive cells (left panel), total numbers of cells (middle panel) and % BrDU-positive cells (right panel). Fields were chosen to contain approximately the same number of cells (as shown in the middle panel) although, as shown by the MTT assay (Fig. 5), ROCK1 knockdown reduced overall cell viability. Values represent means ± SEM (n = 4 independent experiments, in each of which 3 fields of cells were counted for control, ZIPK knockdown and ROCK1 knockdown). *significantly different from control (p < 0.01).
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