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• W2054162629 abstract "Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury. Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury. Cyclic GMP is produced by soluble and receptor guanylate cyclases in response to nitric oxide (NO) 3The abbreviations used are: NO, nitric oxide; BASMCs, bovine aortic smooth muscle cells; 8-CPT-cGMP, 8-para-chlorophenylthio-cGMP; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; KLF, Krüppel-like factor; oligodNT, oligodeoxyribonucleotide; PKG, cGMP-dependent protein kinase; PKN, protein kinase N; PRK, protein kinase C-related kinase; RBD, Rho/Rac-binding domain; ROK, Rho-kinase; RT, reverse transcription; SRF, serum response factor; VASP, vasodilator-stimulated phosphoprotein; VSMCs, vascular smooth muscle cells; siRNA, small interfering RNA; Inr, initiator. 3The abbreviations used are: NO, nitric oxide; BASMCs, bovine aortic smooth muscle cells; 8-CPT-cGMP, 8-para-chlorophenylthio-cGMP; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; KLF, Krüppel-like factor; oligodNT, oligodeoxyribonucleotide; PKG, cGMP-dependent protein kinase; PKN, protein kinase N; PRK, protein kinase C-related kinase; RBD, Rho/Rac-binding domain; ROK, Rho-kinase; RT, reverse transcription; SRF, serum response factor; VASP, vasodilator-stimulated phosphoprotein; VSMCs, vascular smooth muscle cells; siRNA, small interfering RNA; Inr, initiator. and natriuretic peptides, respectively (1Munzel T. Feil R. Mulsch A. Lohmann S.M. Hofmann F. Walter U. Circulation. 2003; 108: 2172-2183Crossref PubMed Scopus (269) Google Scholar). Cellular targets of cGMP include cGMP-dependent protein kinases (PKGs), cGMP-regulated phosphodiesterases, and cGMP-gated ion channels (1Munzel T. Feil R. Mulsch A. Lohmann S.M. Hofmann F. Walter U. Circulation. 2003; 108: 2172-2183Crossref PubMed Scopus (269) Google Scholar). Type I and II PKG are products of different genes and differ in tissue distribution and function (2Lohmann S.M. Vaandrager A.B. Smolenski A. Walter U. De Jonge H.R. Trends Biochem. Sci. 1997; 22: 307-312Abstract Full Text PDF PubMed Scopus (351) Google Scholar). PKG I mediates many cGMP effects on cell proliferation, differentiation, and apoptosis, and PKG I knock-out mice have impaired smooth muscle relaxation, increased platelet aggregation, specific neuronal defects, and a decreased life span (1Munzel T. Feil R. Mulsch A. Lohmann S.M. Hofmann F. Walter U. Circulation. 2003; 108: 2172-2183Crossref PubMed Scopus (269) Google Scholar, 3Pilz R.B. Casteel D.E. Circ. Res. 2003; 93: 1034-1046Crossref PubMed Scopus (246) Google Scholar, 4Bonnevier J. Fassler R. Somlyo A.P. Somlyo A.V. Arner A. J. Biol. Chem. 2004; 279: 5146-5151Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 5Feil R. Hofmann F. Kleppisch T. Reviews in the Neurosciences. 2005; 16: 23-41Crossref PubMed Scopus (71) Google Scholar). Two isoforms of PKG I, α and β, differ in their N-terminal 100 amino acids and are splice variants of the two most 5′ exons of the PKG I gene, which appear to be flanked by separate GC-rich promoters (6Orstavik J. Natarajan V. Tasken K. Jahnsen T. Sandberg M. Genomics. 1997; 42: 311-318Crossref PubMed Scopus (100) Google Scholar). PKG Iα is expressed highly in vascular smooth muscle cells (VSMCs), platelets, and cerebellum, whereas PKG Iβ is prevalent in other parts of the central nervous system, the adrenal gland, and uterus (1Munzel T. Feil R. Mulsch A. Lohmann S.M. Hofmann F. Walter U. Circulation. 2003; 108: 2172-2183Crossref PubMed Scopus (269) Google Scholar, 6Orstavik J. Natarajan V. Tasken K. Jahnsen T. Sandberg M. Genomics. 1997; 42: 311-318Crossref PubMed Scopus (100) Google Scholar). Within a given cell type or tissue, PKG I expression varies greatly, depending on growth conditions. A transient decrease in PKG I mRNA occurs in VSMCs exposed to mitogens (7Tamura N. Itoh H. Ogawa Y. Nakagawa O. Harada M. Chun T.-H. Suga S. Yoshimasa T. Nakao K. Hypertension. 1996; 27: 552-557Crossref PubMed Google Scholar), and some but not all investigators have observed lower PKG Iα expression in actively proliferating, subconfluent VSMC cultures compared with postconfluent cultures (8Cornwell T.L. Soff G.A. Traynor A.E. Lincoln T.M. J. Vascular Res. 1994; 31: 330-337Crossref PubMed Scopus (90) Google Scholar, 9Lin G. Chow S. Lin J. Wang G. Lue T.F. Lin C.S. J. Cell Biochem. 2004; 92: 104-112Crossref PubMed Scopus (21) Google Scholar). In early passage VSMCs and cardiomyocytes and in intact blood vessels, NO or cGMP or cAMP analogs decrease PKG Iα mRNA and protein expression by decreasing transcription; inflammatory cytokines down-regulate PKG Iα in VSMCs by inducing NO synthase and increasing cGMP production (10Soff G.A. Cornwell T.L. Cundiff D.L. Gately S. Lincoln T.M. J. Clin. Invest. 1997; 100: 2580-2587Crossref PubMed Scopus (99) Google Scholar, 11Wollert K.C. Fiedler B. Gambaryan S. Smolenski A. Heineke J. Butt E. Trautwein C. Lohmann S.M. Drexler H. Hypertension. 2002; 39: 87-92Crossref PubMed Scopus (116) Google Scholar, 12Gao Y. Dhanakoti S. Trevino E.M. Wang X. Sander F.C. Portugal A.D. Raj J.U. Am. J. Physiol. 2004; 286: L786-L792Google Scholar, 13Browner N.C. Sellak H. Lincoln T.M. Am. J. Physiol. 2004; 287: C88-C96Crossref PubMed Scopus (50) Google Scholar). After vascular injury in vivo, PKG I expression is transiently reduced in proliferating neointimal VSMCs compared with the normal vessel wall (14Anderson P.G. Boerth N.J. Liu M. McNamara D.B. Cornwell T.L. Lincoln T.M. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2192-2197Crossref PubMed Scopus (69) Google Scholar, 15Sinnaeve P. Chiche J.D. Gillijns H. Van Pelt N. Wirthlin D. Van De W.F. Collen D. Bloch K.D. Janssens S. Circulation. 2002; 105: 2911-2916Crossref PubMed Scopus (57) Google Scholar, 16Melichar V.O. Behr-Roussel D. Zabel U. Uttenthal L.O. Rodrigo J. Rupin A. Verbeuren T.J. Kumar A. Schmidt H.H.H.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16671-16676Crossref PubMed Scopus (89) Google Scholar). During myoblast differentiation, PKG I expression is up-regulated by the transcription factor FoxO1a to orchestrate myoblast fusion (17Bois P.R.J. Brochard V.F. Salin-Cantegrel A.V.A. Cleveland J.L. Grosveld G.C. Mol. Cell. Biol. 2005; 25: 7645-7656Crossref PubMed Scopus (25) Google Scholar). Rho family proteins, including RhoA and Rac1, cycle between an active, GTP-bound state and an inactive, GDP-bound state; their activities are regulated downstream of cell-cell adhesion receptors and various mitogen receptors via guanine nucleotide exchange factors and GTPase-activating proteins (18Fukata M. Kaibuchi K. Nat. Rev. Mol. Cell. Biol. 2001; 2: 887-897Crossref PubMed Scopus (355) Google Scholar, 19Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 20Wennerberg K. Der C.J. J. Cell Sci. 2004; 117: 1301-1312Crossref PubMed Scopus (475) Google Scholar, 21Symons M. Settleman J. Trends in Cell Biology. 2000; 10: 415-419Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Rho proteins regulate actin organization, muscle contractility, cell motility, cell cycle progression, and gene expression (19Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Active RhoA induces actin polymerization and stress fiber formation; it increases the expression of smooth muscle-specific genes through actin-regulated cooperation between serum response factor (SRF) and transcription factors of the myocardin family (22Miano J.M. J. Mol. Cell. Cardiol. 2003; 35: 577-593Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 23Wang D.Z. Olson E.N. Curr. Opin. Genet. Develop. 2004; 14: 558-566Crossref PubMed Scopus (182) Google Scholar). RhoA can also repress gene expression (e.g. RhoA down-regulates transcription of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 and inhibits cytokine-induced transcription of the inducible NO synthase gene), but mechanism(s) of transcriptional inhibition by RhoA remain largely unknown (24Olson M.F. Paterson H.F. Marshall C.J. Nature. 1998; 394: 295-299Crossref PubMed Scopus (409) Google Scholar, 25Adnane J. Bizouarn F.A. Qian Y. Hamilton A.D. Sebti S.M. Mol. Cell Biol. 1998; 18: 6962-6970Crossref PubMed Google Scholar, 26Delarue F.L. Taylor B.S. Sebti S.M. Oncogene. 2001; 20: 6531-6537Crossref PubMed Scopus (31) Google Scholar, 27Muniyappa R. Xu R. Ram J.L. Sowers J.R. Am. J. Physiol. 2000; 278: H1762-H1768Crossref PubMed Google Scholar). PKG I plays a major role in vascular homeostasis and determines vascular responses to NO. Activation of PKG I mediates NO-induced smooth muscle relaxation and inhibition of VSMC proliferation and migration (1Munzel T. Feil R. Mulsch A. Lohmann S.M. Hofmann F. Walter U. Circulation. 2003; 108: 2172-2183Crossref PubMed Scopus (269) Google Scholar). However, little is known about the mechanisms regulating PKG I expression in VSMCs. We found that PKG I expression is controlled by RhoA and Rac1 activity and that RhoA regulation of the PKG Iα promoter is mediated, at least in part, through binding of the Krüppel-like transcription factor KLF4 to Sp1 consensus sites in the proximal promoter. DNA Constructs, Antibodies, and Reagents—The human PKG Iα promoter (positions –430/+35 relative to the transcription start site (6Orstavik J. Natarajan V. Tasken K. Jahnsen T. Sandberg M. Genomics. 1997; 42: 311-318Crossref PubMed Scopus (100) Google Scholar)) was amplified by PCR using genomic DNA from the leukemic cell line HL-60 as a template. This promoter construct does not include the recently described binding sites for FoxO1a or upstream regulatory factors 1/2 (17Bois P.R.J. Brochard V.F. Salin-Cantegrel A.V.A. Cleveland J.L. Grosveld G.C. Mol. Cell. Biol. 2005; 25: 7645-7656Crossref PubMed Scopus (25) Google Scholar, 28Sellak H. Choi C. Browner N. Lincoln T.M. J. Biol. Chem. 2005; 280: 18425-18433Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The PCR product was cloned into the pSVOA luciferase vector (29de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2478) Google Scholar); the pSVOA parent vector was chosen because of low basal luciferase expression. Truncated promoter constructs (–80/+35, –430/+22, and –430/+5) were generated by PCR using appropriate primers. Point mutations in putative Sp1 binding sites were introduced into the full-length promoter using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, with the following oligodeoxynucleotides (oligodNTs; sense strand, underlined letters indicate mutated bases): 5′-CCGCCGCCGCCGCCAAACGAGAAAAAGTTTC-3′(mutated A-site located at +1), 5′-GAAAAAGTTTCGCGGAAAGGCTCAGTGAAAAA-3′ (mutated B-site at +22), 5′-GAGGGGGACGAGGGAAAGGGTCTCAGGGGAG-3′ (mutated C-site at –294), and 5′-TCAGGGGAGGAAGGAAAGCTCTAATTGGTT-3′ (mutated D-site at –272). All PCR products were sequenced. The bacterial expression vector pGEX-mPAK3 (amino acids 65–137) was from R. Cerione (30Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), and the vector for KLF5 (IKLF) was from J. B Lingrel (31Conkright M.D. Wani M.A. Anderson K.P. Lingrel J.B. Nucleic Acids Res. 1999; 27: 1263-1270Crossref PubMed Scopus (143) Google Scholar). Vectors for human KLF4 (GKLF), Rho-related proteins, Rho effectors, and the control plasmid pTK-βGal were described previously (32Chen Z.Y. Shie J.L. Tseng C.C. J. Biol. Chem. 2002; 277: 46831-46839Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 33Gudi T. Chen J.C. Casteel D.E. Seasholtz T.M. Boss G.R. Pilz R.B. J. Biol. Chem. 2002; 277: 37382-37393Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The anti-C-terminal PKG I antibody was from StressGen; an antibody specific for PKG Iβ, antibodies for the Rho isoforms A–C, and antibodies for Sp1 and Sp3 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies specific for Rac1 and glucose-6-phosphate dehydrogenase were from Upstate Biotechnology, Inc., and Sigma, respectively; anti-HA epitope and anti-KLF5 antibodies were from Covance and Orbigen, respectively. Antibodies recognizing human and rat KLF4 and anti-EE epitope antibodies were described previously (34Shie J.L. Chen Z.Y. O'Brien M.J. Pestell R.G. Lee M.E. Tseng C.C. Am. J. Physiol. 2000; 279: G806-G814Crossref PubMed Google Scholar, 35Casteel D.E. Zhuang S. Gudi T. Tang J. Vuica M. Desiderio S. Pilz R.B. J. Biol. Chem. 2002; 277: 32003-32014Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Calpeptin, jasplakinolide, and latrunculin B were from Calbiochem, and 8-para-chlorophenylthio-cGMP (8-CPT-cGMP) was from Biolog. Cell Culture, DNA Transfection, and Reporter Gene Assays—CS54 rat pulmonary artery smooth muscle cells were from A. Rothman (36Rothman A. Kulik T.J. Taubman M.B. Berk B.C. Smith W.J. Nadal-Ginard B. Circulation. 1992; 86: 1977-1986Crossref PubMed Google Scholar), primary bovine aortic smooth muscle cells were from Clonetics (used at passages 3–5), and REF52 rat embryonal fibroblasts were from J. Feramisco. All cells, including HEK 293 cells, were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (33Gudi T. Chen J.C. Casteel D.E. Seasholtz T.M. Boss G.R. Pilz R.B. J. Biol. Chem. 2002; 277: 37382-37393Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In DNA transfection experiments, cells were grown to ∼70% confluence and transfected using Lipofectamine™ Plus (Invitrogen); cells were harvested after 24 h in serum-containing medium, and firefly luciferase and β-galactosidase activities were measured as described previously (33Gudi T. Chen J.C. Casteel D.E. Seasholtz T.M. Boss G.R. Pilz R.B. J. Biol. Chem. 2002; 277: 37382-37393Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). siRNA Transfection—An siRNA for green fluorescent protein (GFP) was from Dharmacon. For RhoA siRNA, we used the oligoribonucleotides 5′-GAAGUCAAGCAUUUCUGUCTT-3′ and 5′-GACAGAAAUGCUUGACUUCTT-3′, which were produced by Qiagen. The sequence for KLF4 siRNA has been described previously (37Chen Z.Y. Tseng C.C. Mol. Pharmacol. 2005; 68: 1203-1213Crossref PubMed Scopus (48) Google Scholar). Each pair of oligoribonucleotides was annealed at a concentration of 20 μm and introduced into cells by transfection with either Oligofectamine™ or Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. Western Blots and PKG Activity Assay—SDS-PAGE and Western blotting were performed as described (33Gudi T. Chen J.C. Casteel D.E. Seasholtz T.M. Boss G.R. Pilz R.B. J. Biol. Chem. 2002; 277: 37382-37393Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Western blots were developed using horseradish peroxidase-coupled secondary antibodies and enhanced chemiluminescence. PKG activity was measured in cell extracts as the difference between Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphorylation in the presence and absence of 3 μm 8-bromo-cGMP; assays were performed in the presence of 100 μm [γ-32PO4]ATP, 300 μm Kemptide, and a 0.3 μm concentration of the specific protein kinase A inhibitor PKI (33Gudi T. Chen J.C. Casteel D.E. Seasholtz T.M. Boss G.R. Pilz R.B. J. Biol. Chem. 2002; 277: 37382-37393Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Quantitative Reverse Transcription-PCR (RT-PCR)—Total cytoplasmic RNA was extracted and subjected to reverse transcription with oligo(dT) primers (35Casteel D.E. Zhuang S. Gudi T. Tang J. Vuica M. Desiderio S. Pilz R.B. J. Biol. Chem. 2002; 277: 32003-32014Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). For the semiquantitative RT-PCR specific for PKG Iα shown in Fig. 1B, the first strand cDNA was amplified with intron-spanning primers generating a 486-bp product as described (35Casteel D.E. Zhuang S. Gudi T. Tang J. Vuica M. Desiderio S. Pilz R.B. J. Biol. Chem. 2002; 277: 32003-32014Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a control for equal mRNA loading. Quantitative RT-PCR was performed using an Mx3000 real time PCR detection system (Stratagene) using IQ™ SYBR Green Supermix (Bio-Rad) according to the manufacturer's protocol with a 0.2 μm concentration of the following primers: 5′-GCGTTCCGGAAGTTCACTAA-3′ (PKG I, sense) and 5′-TTGATGATGCAGCTCTCCTTC-3′ (PKG I, anti-sense); 5′-ACTAACCGTTGGCGAGAGGAAC-3′ (KLF4, sense) and 5′-TGGGATAGCGAGTTGGAAAGG-3′ (KLF4, antisense); 5′-CGTGGTTCACACCCATCACAAAC-3′ (GAPDH, sense) and 5′-GCAAGTTCAACGGCACAGTCAAG-3′ (GAPDH, antisense). DNA was denaturated at 95 °C for 30 s, with annealing and extension occurring at 60 °C for 1 min; each primer pair generated a single product as determined by analyzing melting curves after a 40-cycle control reaction. Standard curves were generated by plotting Ct values versus the amount of input RNA for each primer pair and demonstrated similar amplification efficiencies. Relative changes in mRNA expression were analyzed using the 2–ΔΔCt method (38Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124148) Google Scholar), with GAPDH serving as an internal reference to correct for differences in RNA extraction or reverse transcription efficiencies. Measurement of Rho and Rac Activity—The activation state of Rho was measured using the Rho binding domain of rhotekin (rhotekin-RBD) to isolate Rho·GTP; the amount of RhoA·GTP or RhoB·GTP bound to rhotekin-RBD-coated beads was assessed by Western blotting with RhoA- or RhoB-specific antibodies, respectively (39Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1362) Google Scholar). The activation state of Rac was measured using the Rac/CDC42-binding domain of murine p21-activated kinase-1 (PAK-RBD) and quantitating the isolated Rac·GTP by Western blotting with a Rac1-specific antibody (30Bagrodia S. Taylor S.J. Creasy C.L. Chernoff J. Cerione R.A. J. Biol. Chem. 1995; 270: 22731-22737Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 40Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). Cells were extracted in ice-cold lysis buffer containing 2% Nonidet P-40, 10 mm MgCl2, and protease and phosphatase inhibitors (40Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar, 41Chen J.C. Zhuang S. Nguyen T.H. Boss G.R. Pilz R.B. J. Biol. Chem. 2003; 278: 2807-2818Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Lysates were cleared by centrifugation at 10,000 × g for 2 min and were diluted to 1 mg/ml protein concentration. To 0.8 mg of cell lysate protein was added either 20 μg of rhotekin-RBD or 10 μg of PAK-RBD bound to glutathione-agarose (via glutathione S-transferase tag), and the mixture was incubated with gentle rocking at 4 °C for 60 min. Beads were washed with lysis buffer four times and eluted in SDS-sample buffer for Western blot analysis (41Chen J.C. Zhuang S. Nguyen T.H. Boss G.R. Pilz R.B. J. Biol. Chem. 2003; 278: 2807-2818Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). EMSAs—Nuclear extracts were incubated with 5′-end-labeled double-stranded oligodNT probes and analyzed by nondenaturing PAGE/autoradiography; for supershift experiments, 10 μg of nuclear extract protein were preincubated for 30 min with 1 μg of the indicated antibody (35Casteel D.E. Zhuang S. Gudi T. Tang J. Vuica M. Desiderio S. Pilz R.B. J. Biol. Chem. 2002; 277: 32003-32014Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). OligodNTs corresponding to –3 to +29 relative to the transcription start site of PKG Iα (5′-CCGCCGCCCGAGAAAAAGTTTCGCGGAGGGGC-3′ for upper strand, with putative Sp1 sites A and B in boldface type), were synthesized, annealed, gel-purified, and used as the probe designated Sp1(AB). The Sp1 consensus site oligodNT (5′-ATTCGATCGGGGCGGGGCGAGC-3′) was from Promega, and the oligodNT corresponding to the human Vβ promoter initiator (Inr) sequence was described previously (35Casteel D.E. Zhuang S. Gudi T. Tang J. Vuica M. Desiderio S. Pilz R.B. J. Biol. Chem. 2002; 277: 32003-32014Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Statistical Analysis—The data in bar graphs represent the mean ± S.D. of at least three independent experiments performed in duplicate. Blots, autoradiographs, or photographs represent typical experiments reproduced at least three times with similar results. Statistical analyses were performed using a Student's t test, with a two-tailed value of p < 0.05 considered significant. Cell Density-dependent Expression of PKG I—Cornwell et al. (8Cornwell T.L. Soff G.A. Traynor A.E. Lincoln T.M. J. Vascular Res. 1994; 31: 330-337Crossref PubMed Scopus (90) Google Scholar) observed that PKG I expression in primary rat aortic VSMCs is regulated by cell density with highest levels found in postconfluent cultures. However, others found little differences in PKG expression at different cell densities (9Lin G. Chow S. Lin J. Wang G. Lue T.F. Lin C.S. J. Cell Biochem. 2004; 92: 104-112Crossref PubMed Scopus (21) Google Scholar). We examined PKG I expression at two different cell densities in the rat pulmonary artery smooth muscle cell line CS54, in primary bovine aortic smooth muscle cells (BASMCs), and in REF52 fibroblasts; cells were plated at 0.1–0.2 × 106/10-cm dish or at a 10-fold higher density and cultured for 48 h to generate subconfluent and postconfluent cultures, respectively. As shown in Fig. 1A, PKG I protein levels were about 3-fold higher in postconfluent compared with subconfluent CS54 cultures, with equal protein loading shown by immunoblotting for glucose-6-phosphate dehydrogenase (G6PDH). Similar results were obtained in BASMCs, and an even greater relative increase in PKG I expression was observed in REF52 cells. Like primary VSMCs (10Soff G.A. Cornwell T.L. Cundiff D.L. Gately S. Lincoln T.M. J. Clin. Invest. 1997; 100: 2580-2587Crossref PubMed Scopus (99) Google Scholar), CS54 and REF52 cells express predominantly PKG Iα; using an isotype-specific antibody, we were able to detect low levels of PKG Iβ in CS54 cells, with minimal differences between subconfluent and postconfluent cells (data not shown). Similar to the increased PKG I protein, we found an increase in PKG activity from 413 ± 39 pmol/min/mg in subconfluent cells to 816 ± 104 pmol/min/mg protein in postconfluent CS54 cells (n = 3). Cornwell et al. (8Cornwell T.L. Soff G.A. Traynor A.E. Lincoln T.M. J. Vascular Res. 1994; 31: 330-337Crossref PubMed Scopus (90) Google Scholar) reported comparable PKG activities and differences between subconfluent and postconfluent cultures in primary rat aortic VSMCs. Semiquantitative RT-PCR with PKG Iα-specific primers showed about 3-fold higher PKG Iα mRNA levels in postconfluent CS54 cells compared with subconfluent cells, whereas GAPDH mRNA levels were similar under both conditions (Fig. 1B). To quantitate PKG I mRNA expression more accurately, we used fluorescence-based, real time RT-PCR and measured PKG I relative to GAPDH expression. In CS54 cells and BASMCs, relative PKG I mRNA levels were 2–3-fold higher in postconfluent cells compared with subconfluent cells; in REF52 cells, PKG I mRNA levels increased 10-fold when cells went form a subconfluent to a postconfluent state (Fig. 1C). To determine whether the observed differences in PKG I expression affected cGMP signaling in VSMCs, we examined phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a physiologically important substrate of PKG I in VSMCs (42Smolenski A. Bachmann C. Reinhard K. Hoenig-Liedl P. Jarchau T. Hoschuetzky H. Walter U. J. Biol. Chem. 1998; 273: 20029-20035Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 43Zhuang S. Nguyen G.T. Chen Y. Gudi T. Eigenthaler M. Jarchau T. Walter U. Boss G.R. Pilz R.B. J. Biol. Chem. 2004; 279: 10379-10407Abstract Full Text Full Text PDF Scopus (36) Google Scholar). When subconfluent or postconfluent CS54 cells were treated for 10 min with increasing concentrations of a membrane-permeable cGMP analog, half-maximal VASP phosphorylation on Ser239, the preferred PKG I phosphorylation site, occurred at about 3 μm 8-CPT-cGMP in postconfluent cells, whereas in subconfluent cells, it required >10 μm (Fig. 1D). The left upper panel shows a Western blot developed with a VASP phospho-Ser239-specific antibody; cGMP concentrations of >10 μm are not shown. Note that additional phosphorylation of VASP on Ser157 induces a gel shift with the appearance of a lower mobility species (42Smolenski A. Bachmann C. Reinhard K. Hoenig-Liedl P. Jarchau T. Hoschuetzky H. Walter U. J. Biol. Chem. 1998; 273: 20029-20035Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Equal loading was demonstrated by blotting with an anti-tubulin antibody (Fig. 1D, left lower panel). When CS54 cells were incubated with 10 μm 8-CPT-cGMP, postconfluent cells demonstrated faster kinetics of VASP Ser239 phosphorylation compared with subconfluent cells (Fig. 1D, right upper panel). Thus, increased PKG I expression in postconfluent VSMCs led to faster VASP phosphorylation at lower cGMP concentrations, indicating enhanced cGMP signal transduction efficiency. Cell Density-dependent Regulation of Rho and Rac Activation—In epithelial cell cultures, cell density-dependent signals from cadherin-mediated cell-cell adhesions regulate the activity of Rho family proteins (18Fukata M. Kaibuchi K. Nat. Rev. Mol. Cell. Biol. 2001; 2: 887-897Crossref PubMed Scopus (355) Google Scholar). VSMCs express different types of cadherins than epithelial cells, and to our knowledge, the effect of cell density on the activity of Rho-related proteins in VSMCs has not been determined. We examined the effect of cell density on Rho and Rac activation in CS54 cells, using the GTPase-binding domains of rhotekin or PAK for affinity precipitation of GTP-bound Rho isoforms (RhoA, -B, and -C) or Rac1, respectively (39Ren X.-D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1362) Google Scholar, 40Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). Using a RhoA-specific antibody, we found that RhoA·GTP was about 3 times higher in subconfluent CS54 cells than in postconfluent cells (Fig. 1E, upper panel); cell lysates contained similar amounts of total RhoA (lower panel). RhoB·GTP levels were also significantly higher in subconfluent cells compared with postconfluent cells (data not shown). We determined that RhoA was the predominant Rho isoform expressed in CS54 cells by comparing cell lysate pro" @default.
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• W2054162629 date "2006-06-01" @default.
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• W2054162629 title "Regulation of cGMP-dependent Protein Kinase Expression by Rho and Krüppel-like Transcription Factor-4" @default.
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• W2054162629 doi "https://doi.org/10.1074/jbc.m602099200" @default.
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