Role of endogenous myosin phosphatase inhibitory protein, CPI-17 in vascular smooth muscle

概要

Introduction and aim

Contractile machinery in smooth muscle cells is regulated by myosin light chain (MLC) phosphorylation, which is regulated by the balance between Ca2+/ calmodulin / MLC kinase (MLCK) and MLC phosphatase (MLCP). There are two negatively regulatory signaling for MLCP; one is RhoA/RhoA- associated protein kinase (ROCK)/ myosin binding regulatory protein1(MYPT1) signaling, the other is protein kinase C (PKC)/ CPI-17 signaling. Activation of these Ca2+ independent signaling inhibits MLCP activity which in turn indirectly activates MLCK activity to increase MLC phosphorylation. It is named Ca2+ sensitization of contractile machinery. CPI-17 is a 17kDa protein which can be phosphorylated by PKC mainly at Threonine 38 (T38). The phosphorylated CPI-17 can bind to PP1cδ, a catalyst subunit of MLCP, to inhibit phosphatase activity. However, the detailed function of CPI-17 in vivo is still unclear because of the absence of transgenic mice targeting CPI-17. In this study, I focused on the physiological and pathophysiological function of CPI-17 on blood vessel, using CPI-17 genetically modified mice.

Method

After Dr. Fuji and Dr. Kakuta successfully generated the CPI-17 mutant hetero-mice using clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) technology. I performed western blot, genotyping, immunohistochemistry and sequence method to establish the CPI-17 knock out mice (KO) and mutant CPI-17 mice in which T38 was substituted to　alanine (TA) or glutamate (TE). Then, effects of CPI-17 on vascular contractility in vitro were investigated in isolated thoracic aorta stimulated with different kinds of activator (72.7 mM KCl, 1µM phenylephrine (PE) and 1µM phorbol 12, 13-dibutyrate (PDBu)). The protein expression level of phosphorylated MLC (p-MLC), CPI-17 (p-CPI-17), MYPT1 (p-MYPT1 T853, p-MYPT1 T696) were　detected by western blot. The mean blood pressure (MBP) were tested using the invasive radio telemetry and non-invasive tail-cuff method in vivo to see the function of CPI-17 on maintaining blood pressure. The function of CPI-17 on pathophysiological condition was investigated through carotid artery ligation- induced vascular remodeling model.

Results

Vascular wall thickness of isolated thoracic aorta was not markedly different between WT, KO and TA mice. In terms of mRNA expression, RhoA and ROCK2 of TA were higher than WT and KO mice which showed no significant difference with each other. MLCK mRNA of KO and TA were lower than WT, MYPT1 showed no difference among WT, KO and TA. On physiological or high K+- stimulated condition, the phosphorylation level of MYPT1 and MLC showed no difference among WT, KO and TA, suggesting that the increased RhoA and ROCK2 mRNA may not connect with the contraction ability but compensate other function way which relies on phosphorylation of CPI-17. PDBu- induced contraction was abolished in aorta isolated from KO, TA and TE while induced sustained contraction in WT suggestting that phorbol ester-induced contraction is completely dependent on the T38 phosphorylation site of CPI-17-mediated signaling. TA mutant showed phospho-resistant phenotype. Unfortunately, TE mutant also showed phospho-resistant but not phospho-mimetic phenotype of CPI-17. The decreased expression level of p-MYPT1 at T853 and T696 in KO may also suggest that PDBu has not only an activation activity to induce contraction and phosphorylate CPI-17 and MYPT T853, but also an inhibitory effect on MYPT1 when the function of CPI-17 is depletion or inhibited. Phenylephrine- induced contractility was equivalent in KO and TA animals while it was lower than WT, which indicate that phenylephrine-induced contraction is, at least in part, mediated by phospho-T38-CPI-17 through G- protein/PKC signaling and/or G-protein/RhoA/ROCK.
In vivo experiment, MBP was lower and heart rate was higher in KO and TA compared with WT, suggesting that CPI-17, especially the phosphorylation at T38 of CPI-17 appears to be essential for the physiological maintenance of blood pressure even with an increase in heart rate which occurs as a　compensative action to maintain blood pressure.

Carotid artery ligation which generates disturb flow to induce vascular remodeling was successfully made in WT, KO and TA. KO and TA mice showed increased neointima not only at the position of proximal but also middle part of ligation site in the early stage of vascular remodeling. The mRNA expression of differentiated smooth muscle mark proteins of KO are higher than WT, although there is no significant difference. Taken together, loss of CPI-17 function may play minor role on vascular remodeling at least in ligation model of common carotid artery.

Conclusion

CPI-17 KO and TA constitutively phospho-resistant phenotype were generated for the first time using CRISPR/Cas9 in mice. TE mutant did not show phospho-mimetic phenotype. My findings using these genetically-modified mice demonstrate that phosphorylation of CPI-17 at T38, possibly by protein kinase C, could be important to maintain vascular contractility. CPI-17 is essential for the physiological maintenance of blood pressure. CPI-17 mutant mice would be a useful tool to research the function of CPI-17 on physiological and pathophysiological conditions.

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