Calponin is required for agonist‐induced signal transduction ‐ evidence from an antisense approach in ferret smooth muscle

Abstract

The present study was undertaken to determine whether calponin (CaP) participates in the regulation of vascular smooth muscle contraction and, if so, to investigate the mechanism. By PCR homology cloning, the cDNA sequence of ferret basic (h1) CaP was determined and phosphorothioate antisense and random oligonucleotides were synthesized and introduced into strips of ferret aorta by a chemical loading procedure. Treatment of ferret aorta with CaP antisense oligonucleotides resulted in a decrease in protein levels of CaP to 54 % of that in random sequence‐loaded muscles, but no change in the protein levels of caldesmon (CaD), actin, desmin or extracellular regulated protein kinase (ERK). Contraction in response to phenylephrine or a phorbol ester was significantly decreased in antisense‐treated muscles compared to random sequence‐loaded controls. Neither basal intrinsic tone nor the contraction in response to 51 mm KCl was significantly affected by antisense treatment. During phenylephrine contractions, phospho‐ERK levels increased, as did myosin light chain (LC20) phosphorylation. Phenylephrine‐induced ERK phosphorylation and CaD phosphorylation at an ERK site were significantly decreased by CaP antisense. Increases in myosin light chain phosphorylation were unaffected. The data indicate that CaP plays a significant role in the regulation of contraction and suggest that in a tonically active smooth muscle CaP may function as a signalling protein to facilitate ERK‐dependent signalling, but not as a direct regulator of actomyosin interactions at the myofilament level.

Smooth muscle archvillin: a novel regulator of signaling and contractility in vascular smooth muscle

“…CaP and SmAV colocalize at endogenous levels to the cortices of freshly isolated smooth-muscle cells, after agonist stimulation. Antisense knockdown of SmAV expression decreases PKCactivated contractility and phosphorylation of ERK1/2, signaling effects similar to those observed previously for a CaP knockdown in this system (Je et al, 2001). SmAV, CaP and ERK1/2 localize with F-actin and nonmuscle myosin IIB to PKC-induced 'podosome-like' structures in A7r5 smoothmuscle cells, suggesting that these structures represent sites of CaP-and SmAV-mediated signaling.…”

“…Yeast two-hybrid screening CaP cDNA (Je et al, 2001) was cloned into the bait vector pBD-GAL4 Cam (Stratagene, La Jolla, CA). A HybriZAP ® 2.1 two-hybrid ferret aorta cDNA library was constructed as described (Je et al, 2001), excised from phage and transformed into yeast strain YRG-2 containing the bait vector.…”

“…A HybriZAP ® 2.1 two-hybrid ferret aorta cDNA library was constructed as described (Je et al, 2001), excised from phage and transformed into yeast strain YRG-2 containing the bait vector. Transformants with target plasmids were screened for histidine prototrophy and for β-galactosidase activity according to the manufacturer's instructions (Stratagene).…”

“…Similarly, a chimera of GST and the Cterminal 510 amino acids of bovine supervillin (accession number AF025996; GST-c-BSV) was generated by ligating a partial cDNA into the SmaI/NotI sites of pGEX-6P-3. Recombinant His-tagged ferret h1 CaP was constructed by PCR amplification of the open reading frame (Je et al, 2001) with oligonucleotide primers containing BamHI (sense) and HindIII (antisense) restriction sites. Amplified products were inserted in frame into BamHI/HindIIIdigested pET 30b vector (Novagen, Milwaukee, WI) for bacterial expression and into the pCDNA4/HisMax TOPO vector (Invitrogen Life Technologies, Baltimore, MD) for expression in COS-7 cells.…”

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

“…Furthermore, PD-98059, an ERK1/2 inhibitor, completely abolished the increased responses to ET-1 in both vessels, demonstrating that ERK1/2 is recruited in this abnormal response and suggesting that increased ERK1/2 activity results in augmented contractile responses to ET-1. In this sense, it is known that ERK1/2 phosphorylates caldesmon and calponin, blocking their ability to inhibit the actinmyosin interaction, leading to augmented contractions (22,25,35). In addition, inhibition of ERK attenuates ET-1-induced force development in porcine carotid artery by lowering myosin light chain phosphorylation (5).…”