It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V max . Activity of MP with different substrates also was inhibited by phosphorylation.

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROK␣ isoform. Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates. Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg ؉ -ATPase activity, i.e. similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin. Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rhokinase was stimulated 1.5-2-fold by guanosine 5-O-3-(thio)triphosphate⅐RhoA, whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89. Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30 -50 M) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin. Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.

Although stromal cell-derived factor (SDF)-1a and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1a and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1a and CXCR4. The relationships between clinicopathological features and SDF-1a or CXCR4 expression were then analysed. Stromal cell-derived factor-1a and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1a and nuclear CXCR4 predicts LN metastasis in CRCs.

Pretreatment of alpha-toxin-permeabilized smooth muscle with ATP gamma S (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.

The Regenerating gene (REG) Ia protein, a trophic and/or anti-apoptotic factor, is important in the pathophysiology of gastrointestinal inflammation. Interleukin (IL)-22 is a recently identified cytokine that is suggested to have pivotal roles in inflammatory bowel diseases. We therefore investigated the involvement of the IL-22/REG Ia axis and examined the mechanism of regulation of REG Ia expression by IL-22 stimulation in ulcerative colitis (UC) mucosa. Expression of IL-22, IL-22 receptor 1 (IL-22R1), and REG Ia in UC mucosa was analyzed by real-time RT-PCR and immunohistochemistry. The effects of IL-22 on REG Ia protein expression were examined using a small-interfering RNA for STAT3, an MAPK inhibitor or a PI3K inhibitor. The element responsible for IL-22-induced REG Ia promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay. The expression of IL-22 was enhanced in infiltrating inflammatory cells, and that of IL-22R1 and REG Ia was concurrently enhanced in the inflamed epithelium in UC mucosa. The levels of REG Ia and IL-22 mRNA expression were strongly correlated, and the distributions of REG Ia-and IL-22R1-positive epithelial cells were very similar. IL-22 simulation enhanced the expression of REG Ia protein through STAT3 tyrosine phosphorylation in colon cancer cells. The IL-22-responsive element was located between À142 and À134 in the REG Ia promoter region. REG Ia protein may have a pathophysiological role as a biological mediator for immune cell-derived IL-22 in the UC mucosa.

Interactions of the type 1 phosphatase catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MBP) were compared using affinity columns. In the absence of ATP, MBP bound to dephosphorylated myosin, heavy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not bound. In the presence of ATP, the binding of MBP occurred only with phosphorylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitive inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K(m) for the latter. With myosin, MBP showed a marked increase in kcat, compared to PP1c. ATP did not affect these parameters. To investigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were expressed. These activated PP1c, and activation was maximum at approximately an equimolar ratio. The equimolar mixture of recombinant fragment and PP1c exhibited K(m) values similar to MBP and increased kcat values, compared to PP1c alone. An affinity column was prepared using the recombinant fragment. Phosphorylated HMM and P-LC20 were bound in the presence and absence of ATP. The interaction of P-LC20 was not ATP-dependent. Dephosphorylated HMM did not bind in the presence of ATP. The N-terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the large subunit of MBP contained binding sites for P-LC20 and PP1c.

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Signal transducer and activator of transcription 3 (STAT3) signaling plays roles in inflammation-associated carcinogenesis. Regenerating gene (REG) Ialpha protein, an interleukin (IL)-6-inducible gene, is suggested to be involved in the gastritis-gastric cancer sequence. We investigated the involvement of IL-6/STAT3 signaling in REG Ialpha protein expression and examined whether REG Ialpha protein mediates an anti-apoptotic effect of STAT3 signaling in gastric cancer cells. The effects of IL-6/STAT3 signaling on REG Ialpha protein expression were examined using a STAT3 small interfering RNA system in gastric cancer cells. The element responsible for IL-6-induced REG Ialpha promoter activation was determined by a promoter deletion assay. The anti-apoptotic effects of STAT3 signaling and its induced REG Ialpha protein were examined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphatase nick-end labeling and caspase assay in vitro. Human gastric cancer specimens were analyzed by immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and REG Ialpha protein. IL-6 treatment enhanced the expression of REG Ialpha protein through STAT3 activation in gastric cancer cells. The IL-6-responsive element was determined to lie in the sequence from -142 to -134 of the REG Ialpha promoter region. REG Ialpha protein mediated the anti-apoptotic effects of STAT3 signaling in gastric cancer cells by enhancing Akt activation, Bad phosphorylation and Bcl-xL expression. The expression of REG Ialpha protein was significantly correlated with that of p-STAT3 in gastric cancer tissues. REG Ialpha protein may play a pivotal role in anti-apoptosis in gastric tumorigenesis under STAT3 activation.