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• W2076754423 abstract "The essential role of mechanical signals in regulating the function of living cells is universally observed. However, how mechanical signals are transduced in cells to regulate gene expression is largely unknown. We previously demonstrated that the gene encoding h2-calponin (Cnn2) is sensitively regulated by mechanical tension. In the present study, mouse genomic DNA containing the Cnn2 promoter was cloned, and a nested set of 5′ truncations was studied. Transcriptional activity of the Cnn2 promoter-reporter constructs was examined in transfected NIH/3T3, HEK293, and C2C12 cells for their responses to the stiffness of culture substrate. The results showed significant transcriptional activities of the −1.00- and −1.24-kb promoter constructs, whereas the −0.61-kb construct was inactive. The −1.38-, −1.57-, and −2.12-kb constructs showed higher transcriptional activity, whereas only the −1.57- and −2.12-kb constructs exhibited repression of expression when the host cells were cultured on low stiffness substrate. Internal deletion of the segment between −1.57 and −1.38 kb in the −2.12-kb promoter construct abolished the low substrate stiffness-induced repression. Site-specific deletion or mutation of an HES-1 transcription factor binding site in this region also abolished this repression effect. The level of HES-1 increased in cells cultured under a low tension condition, corresponding to the down-regulation of h2-calponin. h2-Calponin gene expression is further affected by the treatment of cells with Notch inhibitor and activator, suggesting an upstream signaling mechanism. The essential role of mechanical signals in regulating the function of living cells is universally observed. However, how mechanical signals are transduced in cells to regulate gene expression is largely unknown. We previously demonstrated that the gene encoding h2-calponin (Cnn2) is sensitively regulated by mechanical tension. In the present study, mouse genomic DNA containing the Cnn2 promoter was cloned, and a nested set of 5′ truncations was studied. Transcriptional activity of the Cnn2 promoter-reporter constructs was examined in transfected NIH/3T3, HEK293, and C2C12 cells for their responses to the stiffness of culture substrate. The results showed significant transcriptional activities of the −1.00- and −1.24-kb promoter constructs, whereas the −0.61-kb construct was inactive. The −1.38-, −1.57-, and −2.12-kb constructs showed higher transcriptional activity, whereas only the −1.57- and −2.12-kb constructs exhibited repression of expression when the host cells were cultured on low stiffness substrate. Internal deletion of the segment between −1.57 and −1.38 kb in the −2.12-kb promoter construct abolished the low substrate stiffness-induced repression. Site-specific deletion or mutation of an HES-1 transcription factor binding site in this region also abolished this repression effect. The level of HES-1 increased in cells cultured under a low tension condition, corresponding to the down-regulation of h2-calponin. h2-Calponin gene expression is further affected by the treatment of cells with Notch inhibitor and activator, suggesting an upstream signaling mechanism. Mechanical forces have significant effects on various biochemical and genetic processes in living organisms and contribute to multiple physiological regulations and pathological conditions (1Wang N. Tytell J.D. Ingber D.E. Mechanotransduction at a distance. Mechanically coupling the extracellular matrix with the nucleus.Nat. Rev. Mol. Cell Biol. 2009; 10: 75-82Crossref PubMed Scopus (1264) Google Scholar). It is well established that chemical energy can be converted into mechanical forces in living cells by the activity of motor proteins, such as myosin and kinesin/dynein ATPases (2Rayment I. Kinesin and myosin. Molecular motors with similar engines.Structure. 1996; 4: 501-504Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 3Mikhailenko S.V. Oguchi Y. Ishiwata S. Insights into the mechanisms of myosin and kinesin molecular motors from the single-molecule unbinding force measurements.J. R. Soc. Interface. 2010; 7: S295-S306Crossref PubMed Scopus (10) Google Scholar). However, it is much less well understood how mechanical force signals are transduced and converted into chemical signals in cells to regulate biochemical processes and gene expression. The actin cytoskeleton is a dynamic network in eukaryotic cells capable of bearing forces and undergoing rearrangements in adaptation to mechanical changes in the environment, playing essential functions in cellular mechanical properties and responses to mechanical signals (4Kang J. Steward R.L. Kim Y. Schwartz R.S. LeDuc P.R. Puskar K.M. Response of an actin filament network model under cyclic stretching through a coarse grained Monte Carlo approach.J. Theor. Biol. 2011; 274: 109-119Crossref PubMed Scopus (30) Google Scholar). For the dual function of actin cytoskeleton in generating as well as sensing mechanical forces, regulation of actin cytoskeleton is essential for mechanoregulation in eukaryotic cells. Calponin is an actin filament-associated regulatory protein (5Wu K.C. Jin J.P. Calponin in non-muscle cells.Cell Biochem. Biophys. 2008; 52: 139-148Crossref PubMed Scopus (47) Google Scholar). First found in smooth muscle (6Takahashi K. Hiwada K. Kokubu T. Isolation and characterization of a 34,000-dalton calmodulin- and F-actin-binding protein from chicken gizzard smooth muscle.Biochem. Biophys. Res. Commun. 1986; 141: 20-26Crossref PubMed Scopus (261) Google Scholar), three isoforms of calponin (h1, h2, and h3) encoded by homologous genes (CNN1, CNN2, and CNN3) have been identified in vertebrates (7Takahashi K. Nadal-Ginard B. Molecular cloning and sequence analysis of smooth muscle calponin.J. Biol. Chem. 1991; 266: 13284-13288Abstract Full Text PDF PubMed Google Scholar8Nishida W. Kitami Y. Hiwada K. cDNA cloning and mRNA expression of calponin and SM22 in rat aorta smooth muscle cells.Gene. 1993; 130: 297-302Crossref PubMed Scopus (68) Google Scholar, 9Strasser P. Gimona M. Moessler H. Herzog M. Small J.V. Mammalian calponin. Identification and expression of genetic variants.FEBS Lett. 1993; 330: 13-18Crossref PubMed Scopus (116) Google Scholar, 10Applegate D. Feng W. Green R.S. Taubman M.B. Cloning and expression of a novel acidic calponin isoform from rat aortic vascular smooth muscle.J. Biol. Chem. 1994; 269: 10683-10690Abstract Full Text PDF PubMed Google Scholar11Trabelsi-Terzidis H. Fattoum A. Represa A. Dessi F. Ben-Ari Y. der Terrossian E. Expression of an acidic isoform of calponin in rat brain. Western blots on one- or two-dimensional gels and immunolocalization in cultured cells.Biochem. J. 1995; 306: 211-215Crossref PubMed Scopus (48) Google Scholar). h1-Calponin is expressed specifically in differentiated smooth muscle cells (12Winder S.J. Sutherland C. Walsh M.P. Biochemical and functional characterization of smooth muscle calponin.Adv. Exp. Med. Biol. 1991; 304: 37-51Crossref PubMed Scopus (44) Google Scholar). h3-Calponin, also called acidic calponin, is found in smooth muscle cells (10Applegate D. Feng W. Green R.S. Taubman M.B. Cloning and expression of a novel acidic calponin isoform from rat aortic vascular smooth muscle.J. Biol. Chem. 1994; 269: 10683-10690Abstract Full Text PDF PubMed Google Scholar) and neuronal tissues (11Trabelsi-Terzidis H. Fattoum A. Represa A. Dessi F. Ben-Ari Y. der Terrossian E. Expression of an acidic isoform of calponin in rat brain. Western blots on one- or two-dimensional gels and immunolocalization in cultured cells.Biochem. J. 1995; 306: 211-215Crossref PubMed Scopus (48) Google Scholar). In contrast, h2-calponin is present in multiple tissue and cell types, including smooth muscle, lung alveolar cells, endothelial cells, epidermal keratinocytes, fibroblasts, and myeloid leukocytes (9Strasser P. Gimona M. Moessler H. Herzog M. Small J.V. Mammalian calponin. Identification and expression of genetic variants.FEBS Lett. 1993; 330: 13-18Crossref PubMed Scopus (116) Google Scholar, 13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar14Hossain M.M. Smith P.G. Wu K. Jin J.P. Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar, 15Tang J. Hu G. Hanai J. Yadlapalli G. Lin Y. Zhang B. Galloway J. Bahary N. Sinha S. Thisse B. Thisse C. Jin J.P. Zon L.I. Sukhatme V.P. A critical role for calponin 2 in vascular development.J. Biol. Chem. 2006; 281: 6664-6672Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar16Huang Q.Q. Hossain M.M. Wu K. Parai K. Pope R.M. Jin J.P. Role of H2-calponin in regulating macrophage motility and phagocytosis.J. Biol. Chem. 2008; 283: 25887-25899Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Calponin binds F-actin with high affinity and inhibits the actin-activated myosin ATPase (17Winder S.J. Walsh M.P. Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation.J. Biol. Chem. 1990; 265: 10148-10155Abstract Full Text PDF PubMed Google Scholar18Abe M. Takahashi K. Hiwada K. Effect of calponin on actin-activated myosin ATPase activity.J. Biochem. 1990; 108: 835-838Crossref PubMed Scopus (129) Google Scholar, 19Horiuchi K.Y. Chacko S. The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.Biochem. Biophys. Res. Commun. 1991; 176: 1487-1493Crossref PubMed Scopus (68) Google Scholar20Winder S.J. Allen B.G. Fraser E.D. Kang H.M. Kargacin G.J. Walsh M.P. Calponin phosphorylation in vitro and in intact muscle.Biochem. J. 1993; 296: 827-836Crossref PubMed Scopus (90) Google Scholar) and motor (12Winder S.J. Sutherland C. Walsh M.P. Biochemical and functional characterization of smooth muscle calponin.Adv. Exp. Med. Biol. 1991; 304: 37-51Crossref PubMed Scopus (44) Google Scholar, 21Shirinsky V.P. Biryukov K.G. Hettasch J.M. Sellers J.R. Inhibition of the relative movement of actin and myosin by caldesmon and calponin.J. Biol. Chem. 1992; 267: 15886-15892Abstract Full Text PDF PubMed Google Scholar, 22Haeberle J.R. Calponin decreases the rate of cross-bridge cycling and increases maximum force production by smooth muscle myosin in an in vitro motility assay.J. Biol. Chem. 1994; 269: 12424-12431Abstract Full Text PDF PubMed Google Scholar) activities. Extensive biochemical and biophysical studies have demonstrated that calponin regulates the function of actin filaments to modify smooth muscle contractility and non-muscle cell motility. For its expression in multiple tissue and cell types, the function and regulation of h2-calponin is of broad biological and medical significances. h2-Calponin stabilizes actin filaments and inhibits actin cytoskeleton-related cellular functions, such as cytokinesis, migration, and phagocytosis (13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 14Hossain M.M. Smith P.G. Wu K. Jin J.P. Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar, 16Huang Q.Q. Hossain M.M. Wu K. Parai K. Pope R.M. Jin J.P. Role of H2-calponin in regulating macrophage motility and phagocytosis.J. Biol. Chem. 2008; 283: 25887-25899Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The expression of h2-calponin gene and the degradation of h2-calponin protein are both regulated by mechanical tension in the cytoskeleton (13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 14Hossain M.M. Smith P.G. Wu K. Jin J.P. Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar). In response to tension changes, proteolysis of h2-calponin provides rapid structural and functional modifications in the actin cytoskeleton, whereas its gene regulation conveys chronic and sustained alterations. The regulation of h2-calponin gene expression may be studied as a representative for understanding the mechanisms of mechanoregulation in living cells. In a previous study, we demonstrated that h2-calponin protein and mRNA was significantly decreased in NIH/3T3 cells when cytoskeleton tension was reduced after blebbstatin inhibition of myosin II motor function (14Hossain M.M. Smith P.G. Wu K. Jin J.P. Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar). In contrast to the endogenous h2-calponin gene, transfective expression of h2-calponin cDNA under the control of CMV promoter was not regulated by mechanical tension (13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This observation suggests that transcriptional control is a primary regulation for the mechanoregulation of h2-calponin gene expression. The promoter of CNN2 gene therefore provides a novel experimental system to investigate how mechanical force signal is transduced in the regulation of gene transcription (23Shivashankar G.V. Mechanosignaling to the cell nucleus and gene regulation.Annu. Rev. Biophys. 2011; 40: 361-378Crossref PubMed Scopus (125) Google Scholar). In the present study, we characterized the transcriptional activity of h2-calponin gene promoter for its mechanoregulation. We cloned mouse genomic DNA containing the Cnn2 promoter, constructed reporter genes, and analyzed transcriptional activity in several mammalian cell types that express endogenous h2-calponin under mechanoregulation. Using cell culture substrate of different stiffness, transcriptional activities of truncation, deletion, and site-specific mutation promoter constructs were studied to identify an HES-1 (hairy and enhancer of split 1) cis-regulatory element downstream of the Notch signaling pathway. As described previously, NIH/3T3 cells (ATCC CRL1658) were cultured in plastic dishes statically in an attached monolayer or under continuous vibration to prevent attachment to the dishes (26Hossain M.M. Hwang D.Y. Huang Q.Q. Sasaki Y. Jin J.P. Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation.Am. J. Physiol. Cell Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). After cultured at 37 °C in 5% CO2 for 3 days, the cells were washed with PBS, and total cellular protein was extracted for SDS-PAGE and Western blotting analysis. Human embryonic kidney cell line HEK293 (ATCC, CRL1573) and mouse fibroblast line NIH/3T3 were cultured in Dulbecco's modified Eagle's medium containing fetal bovine serum (10%), penicillin (100 IU/ml) and streptomycin (50 IU/ml) at 37 °C in 5% CO2. Monolayer cultures of cells at ∼70% confluence were trypsinized and passed at 1:10 ratio for use in experiments. Mouse skeletal myoblast line C2C12 (ATCC, CRL1772) was cultured with the same media conditions and trypsinized at ∼50% confluence to pass at 1:5 ratio for use in experiments. To examine the effect of substrate stiffness-dependent cytoskeleton tension on h2-calponin gene expression, HEK293, NIH/3T3, and C2C12 cells were seeded on a thin layer (45 × 50 mm, ∼100-μm thickness) of polyacrylamide gel of different stiffness. Hard (16% gel with an acrylamide:bisacrylamide ratio of 19:1, Ef = ∼75 kilopascal) and soft (5% gel with an acrylamide:bisacrylamide ratio of 500:3, Ef = ∼1 kilopascal) gel substrates were prepared and covalently coated with 0.2 mg/ml type I collagen as described previously (13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Wang Y.L. Pelham Jr., R.J. Preparation of a flexible, porous polyacrylamide substrate for mechanical studies of cultured cells.Methods Enzymol. 1998; 298: 489-496Crossref PubMed Scopus (301) Google Scholar, 25Engler A. Bacakova L. Newman C. Hategan A. Griffin M. Discher D. Substrate compliance versus ligand density in cell on gel responses.Biophys. J. 2004; 86: 617-628Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). After 3 days of culture, the cells were harvested using a cell scraper, washed three times with PBS, and processed for analyses. SDS-PAGE and Western blotting were carried out as previously described (13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) to examine the expression of endogenous h2-calponin in the cultured cells. The monolayer cells were directly lysed in SDS-PAGE sample buffer containing 2% SDS and analyzed using 12% gel in Laemmli buffer system with an acrylamide:bisacrylamide ratio of 29:1. After electrophoresis, the gels were fixed and stained with Coomassie Blue R-250 to verify sample integrity and normalize protein concentration. Protein bands in unfixed duplicate gels were electrically transferred to nitrocellulose membrane for Western blotting with a rabbit antiserum, RAH2, raised against mouse h2-calponin (26Hossain M.M. Hwang D.Y. Huang Q.Q. Sasaki Y. Jin J.P. Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation.Am. J. Physiol. Cell Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar) or a monoclonal antibody against HES-1 (7H11, from Abnova). The calponin or HES-1 band recognized by the first antibody was revealed using alkaline phosphatase-labeled anti-rabbit IgG or anti-mouse IgG second antibody (Santa Cruz Biotechnology) and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chromogenic substrate reaction. From the screening of a mouse genomic library with a mouse h2-calponin cDNA probe as described previously (16Huang Q.Q. Hossain M.M. Wu K. Parai K. Pope R.M. Jin J.P. Role of H2-calponin in regulating macrophage motility and phagocytosis.J. Biol. Chem. 2008; 283: 25887-25899Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), lambda phage clones containing mouse Cnn2 gene (NC_000076.5) were isolated. A 5,058-bp BamHI restriction fragment containing the promoter of Cnn2 gene (−3,765 to +1,292) was subcloned into plasmid vectors. The region of −3,765 to +6 was further subcloned as a BamHI-SmaI fragment into the pcDNA3.1(+)/chloramphenical acetyltransferase (CAT) 3The abbreviations used are:CATchloramphenical acetyltransferaseDAPTN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl esterEfelastic forcePEITCphenethyl isothiocyanateRXRvitamin D receptor/retinoic acid X receptor. plasmid vector (Invitrogen) to replace the CMV promoter in front of the CAT coding sequence. chloramphenical acetyltransferase N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester elastic force phenethyl isothiocyanate vitamin D receptor/retinoic acid X receptor. After demonstrating transcriptional activity of the −3.77-kb mouse Cnn2 promoter-CAT reporter construct, we constructed six 5′ serial truncation clones to map the cis-regulatory elements. As illustrated in Fig. 1, −2.12-kb (−2,115 to +6) and −0.61-kb (−611 to +6) clones were constructed from the −3.77-kb clone by restriction enzyme digestion-generated deletions. Four other 5′ truncation Cnn2 promoter constructs, −1.57 kb (−1,572 to +6), −1.38 kb (−1,380 to +6), −1.24 kb (−1,244 to +6), and −1.00 kb (−1,002 to +6), were constructed using PCR-generated DNA fragments from the −3.77-kb template. A common 3′ primer located in the CAT coding sequence was used to pair with different 5′ primers to generate these constructions. The primer pairs introduced a BamHI site at the 5′-end and a NotI restriction site at the 3′-end for unidirectional cloning of the PCR products into pcDNA3.1(+)/CAT reporter plasmid. A −1,572 to −1,380 internal deletion (−1.57-kb/−1.38-kb deletion) was constructed in the −2.12-kb Cnn2 promoter-reporter gene using recombinant PCR. Two oligonucleotide primers with complementary 5′ sequences were designed for use with flanking primers in the first PCR to amplify the −2,115 to −1,572 and −1,380 to +6 segments of the Cnn2 promoter (Fig. 1). A recombinant DNA fragment was generated by joining the two first PCR products in the second PCR. The internal deletion promoter construct was unidirectionally cloned into pcDNA3.1(+)/CAT reporter plasmid as a BamHI-NotI fragment. The 5′ truncation and internal deletion constructs were verified by restriction enzyme mapping and DNA sequencing. HEK293 cells were seeded in 24-well plates at 8 × 104 cells/well and cultured for 16 h. For each transfection, 0.04 pmol of the promoter-reporter plasmid was used with liposome reagent (Turbofect, Fermentas) at a 1:4 DNA/liposome (μg/μl) ratio. pEGFP-N1 plasmid encoding GFP under CMV promoter was co-transfected at a molar ratio of 1:20 versus the Cnn2 promoter construct to indicate the efficiency of transfection. DNA and liposome reagent were mixed in 100 μl of DMEM, incubated at room temperature for 15 min, and added to the cell culture medium. After 6 h, the transfection medium was replaced with fresh medium for continuing culture of 24–30 h. NIH/3T3 and C2C12 cells were transfected similarly. 1 × 105 or 5 × 104 cells/well, respectively, were seeded in 12-well plates. 0.10 pmol of DNA was used for both NIH/3T3 and C2C12 cells at DNA/liposome ratios of 1:2 and 1:4, respectively (μg/μl). The cultures were examined using an inverted epifluorescence microscope for GFP-positive cells to evaluate the efficiency of transfection. The cells were then washed three times with PBS and lysed for use either directly or after short frozen storage at −80 °C in the quantification of reporter gene expression. For every promoter construct, three or more independent transfection experiments were performed to verify the results. To establish stable transfections, Cnn2 promoter-reporter plasmid DNA was linearized with restriction enzyme cleavage at the 5′ flanking BamHI (for the −1.57-, −1.38-, −1.24-, and −1.00-kb constructs) or AflII (for the −2.12-kb construct) site. 1 × 106 HEK293 cells were plated on a 100-mm dish for 16 h before the incubation with 0.2 μg of recombinant plasmid DNA mixed with 0.8 μl of liposome reagent for 6 h. After 24 h, the medium was replaced with fresh medium containing G418 at a concentration predetermined to effectively kill untransfected cells in 2 weeks. Cultured in the selection medium for 10–14 days, single colonies of surviving and proliferating cells were picked manually as described previously (26Hossain M.M. Hwang D.Y. Huang Q.Q. Sasaki Y. Jin J.P. Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation.Am. J. Physiol. Cell Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar), expanded, verified using PCR for the presence of the CAT reporter gene, examined using Western blot for the normal expression of endogenous h2-calponin, and studied for transcriptional activity of the Cnn2 promoter constructs. At least three original stable transfection clones expressing endogenous h2-calponin at a level similar to that of nontransfected control cells were studied for each promoter construct. To map the 5′-upstream region of Cnn2 promoter for cis-regulatory elements responsive to mechanoregulation, the stable transfected cell clones were cultured on polyacrylamide gel substrates of high or low stiffness as described above. After 72 h of culture, samples were collected to examine the expression of endogenous h2-calponin using Western blot and transcriptional activity of the Cnn2 promoter-reporter constructs. The results were summarized from at least three original stable transfection clones that have similar normal level expression of endogenous h2-calponin. CAT-ELISA was performed as previously described (27Huang Q.Q. Jin J.P. Preserved close linkage between the genes encoding troponin I and troponin T, reflecting an evolution of adapter proteins coupling the Ca2+ signaling of contractility.J. Mol. Evol. 1999; 49: 780-788Crossref PubMed Scopus (26) Google Scholar). Briefly, the concentration of cellular protein extracts was determined using Bradford assay and further normalized by SDS-PAGE gel densitometry. CAT expression of the Cnn2 promoter-reporter constructs was examined with a sandwich ELISA method using the reagent kit from Roche Applied Science. 100 μg of total protein extract of the transfected cells was dissolved in 100 μl of reaction buffer and loaded to each assay well of a 96-well plate. The samples were incubated at 37 °C for 2 h followed by standard ELISA steps, including washes, incubations with anti-CAT antibody and horseradish peroxidase-labeled second antibody, and substrate-enhancer reaction. A415 nm of each assay well was monitored at a series of time points with reference wavelength of 655 nm using an automated microplate reader (Bio-Rad Benchmark). Triplicate wells were examined for each sample. The transcriptional activity of the promoter-reporter constructs was normalized for transfection efficiency determined as the percentage of cells positive for the co-transfective expression of GFP. Absorbance data of soft gel cultures were compared with that of corresponding hard gel cultures to determine the percentage of down-regulation of each promoter construct. Bioinformatic analysis of transcription factor binding sites was performed using the Genomatix suite software (Genomatix Software GmbH, Munich, Germany). The sequence of the −2.12-kb promoter segment of mouse Cnn2 gene was used as the target sequence for identification of putative transcription factor recognition sites using the MatInspector Version 8.0.5 program (28Cartharius K. Frech K. Grote K. Klocke B. Haltmeier M. Klingenhoff A. Frisch M. Bayerlein M. Werner T. MatInspector and beyond. Promoter analysis based on transcription factor binding sites.Bioinformatics. 2005; 21: 2933-2942Crossref PubMed Scopus (1637) Google Scholar). The parameters used were the standard (0.75) core similarity and the optimized matrix similarity. Transcription factor binding sites found in the −1,572 to −1,380 and −1,380 to −1,244 regions were considered for potential correlation to mechanoregulation. A deletion from −1,438 to −1,423 was made in the −2.12-kb construct using recombinant PCR as described above for the −1.57 kb/−1.38 kb deletion. An HES-1 N-box mutation was made in the −2.12-kb construct using recombinant PCR, converting the −1,431 to −1,425 region of the Cnn2 promoter from CACGAG to TCTAGA (a new XbaI site was introduced for easy identification). K562 human myelogenous leukemia cells (ATCC, CCL-243) that were growing in culture nonadherently were employed to study the role of Notch signaling in regulating h2-calponin expression. The effects of a Notch inhibitor, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), and a Notch activator, phenethyl isothiocyanate (PEITC), were examined. Cells were seeded at 2 × 105/well in a 12-well culture plate in 1 ml of Iscove's medium containing 10% fetal bovine serum and various concentrations of the drug. The solvent control for DAPT was DMF at a concentration of 0.04%, and for PEITC was Me2SO at a concentration of 0.05%. Cells were cultured for 72 h, collected, and centrifuged at 500 × g for 5 min to remove the media. The cell pellets were washed with PBS for three times and lysed in SDS-PAGE sample buffer for Western blotting as described above. Densitometry analysis of SDS gels and Western blots were performed using National Institutes of Health Image software (version 1.61) on digital images scanned at 600 dpi. The quantitative data are presented as means ± S.D. unless noted in the figure legend. Statistical analysis was done using the Microsoft Excel or Origin software. The significance of difference was established as p < 0.05. We previously reported that h2-calponin is expressed in smooth muscle and multiple non-muscle tissue and cell types including epithelial, endothelial, fibroblast, and myeloid blood cells (13Hossain M.M. Crish J.F. Eckert R.L. Lin J.J. Jin J.P. h2-Calponin is regulated by mechanical tension and modifies the function of actin cytoskeleton.J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar14Hossain M.M. Smith P.G. Wu K. Jin J.P. Cytoskeletal tension regulates both expression and degradation of h2-calponin in lung alveolar cells.Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar, 15Tang J. Hu G. Hanai J. Yadlapalli G. Lin Y. Zhang B. Galloway J. Bahary N. Sinha S. Thisse B. Thisse C. Jin J.P. Zon L.I. Sukhatme V.P. A critical role for calponin 2 in vascular development.J. Biol. Chem. 2006; 281: 6664-6672Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar16Huang Q.Q. Hossain M.M. Wu" @default.
• W2076754423 created "2016-06-24" @default.
• W2076754423 creator A5035485086 @default.
• W2076754423 creator A5056424368 @default.
• W2076754423 creator A5067036313 @default.
• W2076754423 creator A5068088876 @default.
• W2076754423 creator A5076463740 @default.
• W2076754423 creator A5081586533 @default.
• W2076754423 date "2014-01-01" @default.
• W2076754423 modified "2023-10-17" @default.
• W2076754423 title "Mechanoregulation of h2-Calponin Gene Expression and the Role of Notch Signaling" @default.
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