Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis
2008; Elsevier BV; Volume: 283; Issue: 38 Linguagem: Inglês
10.1074/jbc.m801163200
ISSN1083-351X
AutoresQi-Quan Huang, M. Moazzem Hossain, Kaichun Wu, Kakoli Parai, Richard M. Pope, Jian‐Ping Jin,
Tópico(s)Galectins and Cancer Biology
ResumoThe actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neoR cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neoR cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions. The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neoR cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neoR cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions. Leukocytes are mobile cells and their actin cytoskeleton plays a central role in the locomotion, transmigration, and phagocytosis. These activities are essential for the function of myeloid cells, including neutrophils, monocytes, and macrophages, in defensive and autoimmune responses (1Jones G.E. J. Leukocyte Biol. 2000; 68: 593-602PubMed Google Scholar). Despite the significant biological and medical importance, the regulation of actin cytoskeleton in myeloid cells is not well understood. Calponin is an actin filament-associated protein of 34–37 kDa (292–330 amino acids) found in smooth muscle (2Takahashi K. Hiwada K. Kobuku T. Biochem. Biophys. Res. Commun. 1986; 141: 20-26Crossref PubMed Scopus (261) Google Scholar) as well as non-muscle cells (3Hossain M.M. Crish J.F. Eckert R.L. Lin J.J.-C. Jin J.-P. J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,4Hossain M.M. Smith P.G. Wu K. Jin J.-P. Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar). Through high affinity binding to F-actin, calponin inhibits the actin-activated myosin MgATPase (5Winder S. Walsh M.P. J. Biol. Chem. 1990; 265: 10148-10155Abstract Full Text PDF PubMed Google Scholar, 6Abe M. Takahashi K. Hiwada K. J. Biochem. (Tokyo). 1990; 108: 835-838Crossref PubMed Scopus (129) Google Scholar, 7Horiuchi K.Y. Chacko S. Biochem. Biophys. Res. Commun. 1991; 176: 1487-1493Crossref PubMed Scopus (68) Google Scholar, 8Winder S.J. Allen B.G. Fraser E.D. Kang H.-M. Kargacin G.J. Walsh M.P. Biochem. J. 1993; 296: 827-836Crossref PubMed Scopus (90) Google Scholar) and motor activity (9Walsh M.P. Biochem. Cell Biol. 1991; 69: 771-800Crossref PubMed Scopus (122) Google Scholar, 10Shirinsky V.P. Birynkov K.G. Hettasch J.M. Sellers J.R. J. Biol. Chem. 1992; 267: 15886-15892Abstract Full Text PDF PubMed Google Scholar, 11Haeberle J.R. J. Biol. Chem. 1994; 269: 12424-12431Abstract Full Text PDF PubMed Google Scholar). The association of calponin with actin filaments and its regulatory function have led to a model wherein calponin may represent a thin filament regulatory mechanism modulating smooth muscle contraction (12Allen B.G. Walsh M.P. Trends Biochem. Sci. 1994; 19: 362-368Abstract Full Text PDF PubMed Scopus (162) Google Scholar). Three isoforms of calponin (h1, h2, and acidic) have been identified in higher vertebrates as the products of three homologous genes (13Takahashi K. Nadal-Ginard B. J. Biol. Chem. 1991; 266: 13284-13288Abstract Full Text PDF PubMed Google Scholar, 14Nishida W. Kitami Y. Hiwada K. Gene (Amst.). 1993; 130: 297-302Crossref PubMed Scopus (68) Google Scholar, 15Strasser P. Gimona M. Moessler H. Herzog M. Small J.V. FEBS Lett. 1993; 330: 13-18Crossref PubMed Scopus (116) Google Scholar, 16Applegate D. Feng W. Green R.S. Taubman M.B. J. Biol. Chem. 1994; 269: 10683-10690Abstract Full Text PDF PubMed Google Scholar, 17Trabelsi-Terzidis H. Fattoum A. Represa A. Dessi F. Ben-Ari Y. der Terrossian E. Biochem. J. 1995; 306: 211-215Crossref PubMed Scopus (48) Google Scholar). The three isoforms of calponin have distinct theoretical isoelectric points (pI values): H1-calponin is basic (pI 9.4), h2-calponin is near neutral (pI 7.5), whereas the acidic calponin has a lower pI of 5.2. H1-calponin is the predominant isoform specifically expressed in differentiated smooth muscle cells and its role in regulating smooth muscle contractility (9Walsh M.P. Biochem. Cell Biol. 1991; 69: 771-800Crossref PubMed Scopus (122) Google Scholar) is a focus of ongoing research. The majority of previous structural and functional studies of calponin were obtained from chicken gizzard calponin that is equivalent to the mammalian h1-calponin. The acidic calponin has been found in smooth muscle (16Applegate D. Feng W. Green R.S. Taubman M.B. J. Biol. Chem. 1994; 269: 10683-10690Abstract Full Text PDF PubMed Google Scholar) and brain (17Trabelsi-Terzidis H. Fattoum A. Represa A. Dessi F. Ben-Ari Y. der Terrossian E. Biochem. J. 1995; 306: 211-215Crossref PubMed Scopus (48) Google Scholar) and its function remains to be investigated. H2-calponin is found in smooth muscle and certain non-muscle cells (3Hossain M.M. Crish J.F. Eckert R.L. Lin J.J.-C. Jin J.-P. J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 4Hossain M.M. Smith P.G. Wu K. Jin J.-P. Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar, 18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). We previously demonstrated that h2-calponin is expressed at higher levels in developing and remodeling smooth muscles and its overexpression inhibited the rate of cell proliferation (18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). We further found that h2-calponin is expressed in epidermal keratinocytes, lung alveolar epithelial cells, and fibroblasts and plays a role in stabilizing the actin cytoskeleton. The expression and degradation of h2-calponin are both under the regulation of cytoskeletal tension built by myosin motors (3Hossain M.M. Crish J.F. Eckert R.L. Lin J.J.-C. Jin J.-P. J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 4Hossain M.M. Smith P.G. Wu K. Jin J.-P. Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar). The function of h2-calponin in regulating the actin cytoskeleton of non-muscle cells suggests its role in multiple cellular activities. In the present study, we found significant levels of h2-calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed through embryonic stem (ES) 2The abbreviations used are: ES cellembryonic stem cellCnn2mouse h2-calponin geneFBSfetal bovine serumFITCfluorescein isothiocyanatemAbmonoclonal antibodyPBSphosphate-buffered salineDMEMDulbecco's modified Eagle's mediumTRITCtetramethylrhodamine isothiocyanate. cell gene targeting (19Thomas K.R. Capecchi M.R. Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1826) Google Scholar). Germ line transmission of the Cnn2-flox-neo allele was successful in chimeric mice from two independent clones of targeted embryonic stem cells. The insertion of the neoR selection cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the neoR cassette by FLP1 recombinase completely rescued the knockdown effect. The Cre recombinase-induced deletion of exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice had reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free cells showed a higher rate of proliferation and faster migration than that of wild type cells, consistent with a faster migration of blood myeloid cells into tissues. The h2-calponin-free macrophages demonstrated reduced spreading in adherent culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased phagocytotic activity, suggesting a novel mechanism to regulate macrophage function. embryonic stem cell mouse h2-calponin gene fetal bovine serum fluorescein isothiocyanate monoclonal antibody phosphate-buffered saline Dulbecco's modified Eagle's medium tetramethylrhodamine isothiocyanate. All animal procedures were approved by the Institutional Animal Care and Use Committees and were conducted in accordance with the Guiding Principles in the Care and Use of Animals, as approved by the Council of the American Physiological Society. Cloning of Mouse Cnn2 Genomic DNA—From a 129SvJ strain mouse genomic DNA library in λ DASHII phage vector (20Gao J. Hwang J.M. Jin J.-P. Biochem. Biophys. Res. Commun. 1996; 218: 292-297Crossref PubMed Scopus (21) Google Scholar, 21Huang Q.-Q. Chen A. Jin J.-P. Gene (Amst.). 1999; 229: 1-10Crossref PubMed Scopus (45) Google Scholar), clones bearing genomic DNA segments containing the h2-calponin gene were isolated using 32P-labeled mouse h2-calponin cDNA probe. The plating of λ phage, making of nylon membrane replicas, hybridization, and autoradiography were carried out as described previously (21Huang Q.-Q. Chen A. Jin J.-P. Gene (Amst.). 1999; 229: 1-10Crossref PubMed Scopus (45) Google Scholar). The plaque-purified positive phages were amplified in XL-1B MFA P2 Escherichia coli and purified by centrifugation through CsCl density gradients. The recombinant phage DNA was isolated by phenol/chloroform extraction and subcloned into plasmid vectors as overlapping restriction endonuclease fragments. Restriction mapping, Southern analysis, and partial sequencing were carried out to verify the cloned Cnn2 genomic DNA in comparison with the mouse genomic DNA sequence in the data base (Gene ID MGI: 105093). Development of H2-Calponin Gene Knock-out Mice—A mouse Cnn2-targeted conditional mutagenesis construct was generated using the pPNT4 vector (22Conrad M. Brielmeier M. Wurst W. Bornkamm G.W. BioTechniques. 2003; 34 (1140): 1136-1138Crossref PubMed Scopus (20) Google Scholar), provided by Dr. Marcus Conrad, Institute of Clinical Molecular Biology and Tumor Genetics GSF-Research Centre for Environment and Health, Germany). In the Cnn2 gene targeting construct, two loxP sequences were inserted in intron 1 and intron 2 in tandem orientation to allow Cre recombinase-catalyzed deletion of exon 2. The deletion of exon 2 not only removes a portion of the coding sequence but also results in a reading frameshift in the downstream mRNA after codon number 21. The shifted reading frame is terminated by a stop codon after encoding 11 missense amino acids. This Cre-mediated deletion of exon 2 can be induced in whole animals as well as in tissues or in cultured cells. A neoR cassette adjacent to the downstream loxP sequence as constructed in the pPNT4 vector was inserted into intron 2 of the Cnn2 targeting construct for neomycin selection of the transfected ES cells. The neoR cassette is flanked by two frt sequences and can be deleted by FLP1-catalyzed recombination (23Meyers E.N. Lewandoski M. Martin G.R. Nat. Genet. 1998; 18: 136-141Crossref PubMed Scopus (885) Google Scholar). This mechanism allows the removal of the neoR cassette after establishing the targeted loxP mutagenesis to avoid the effect of neoR insertion on h2-calponin expression. The induction of neoR removal can be done in whole animals, tissues, or cells. Long flanking arms (4.6 and 5.5 kb) were placed in the gene targeting construct to provide sufficient regions for homologous recombination. Transfection of mouse HM-1 ES cells (x,y) with the h2-calponin gene targeting DNA construct using electroporation was carried out at the Northwestern University Transgenic and Targeted Mutagenesis Laboratory. Colonies of the transfected ES cells were selected by the acquisition of neomycin resistance conferred by the gene targeting construct. Genomic DNA from the drug-resistant ES cell colonies was extracted by protease K digestion and screened by Southern blotting using cloned 5′- and 3′-flanking genomic DNA probes for the homologous recombination generated change of BamHI restriction pattern. Ten μg each of the ES cell genomic DNA was digested by BamHI, separated by 0.8% agarose gel electrophoresis, and transferred to the nylon membrane by capillary action using standard Southern blotting method. The membrane was prehybridized at 55 °C for 2 h in 0.25 m Na2HPO4, 14 mm H3PO4, 1 mm EDTA, 1% bovine serum albumin, 5% SDS, 0.1 mg/ml mechanically sheared salmon sperm DNA, 20% formamide. The 5′ and 3′ DNA probes were labeled with [32P]dCTP, heat-denatured, and added to the rolling hybridization flask together with 5% (w/v) dextran sulfate for incubation at 55 °C for 16 h. The membrane was then washed repeatedly by 40 mm sodium phosphate buffer containing 1% SDS and 1 mm EDTA to gradually reach 60 °C and examined by autoradiography. Two original Cnn2-targeted mouse ES cell clones identified by Southern screening, 21C7 and 21H2, were used to produce chimeric mice. The blastocyst injection and embryo re-implantation were carried out at the Northwestern University Gene Targeting and Transgenic Core Facility. The 129SvJ-originated (albino) ES cells are injected into C57BL/6 (black) mouse blastocysts to produce chimeras. High chimerism males from the two targeted ES cell lines were mated with C57BL/6 females to test germ line transmission. The ES cell-originated offspring was first selected by the brown coat color in contrast to the pure C57BL/6 black littermates. The presence of the targeted Cnn2 allele in the ES cell-originated pups was then genotyped by PCR on genomic DNA extracted from tail biopsies. Two pairs of PCR primers were designed to identify the presence of the upstream loxP and the neoR cassette, respectively. Mice bearing the targeted Cnn2 allele were selected to mate with C57BL/6 for 7 to 9 generations to obtain a uniformed genetic background. Disruption of the h2-calponin gene through deletion of the exon 2 region was obtained by crossing the Cnn2-flox mouse line with the Zp3-cre mouse line (The Jackson Laboratory) that expresses Cre recombinase in the female germ line. Removal of the neoR cassette inserted in intron 2 was achieved by crossing the Cnn2-flox-neo line with a Gt(ROSA)26Sor-FLP transgenic mouse line (The Jackson Laboratory) that expresses FLP1 recombinase in most tissue types, including the developing germ line. SDS-PAGE and Western Blotting—Representative tissue samples were obtained from adult (4–5 months old) wild type and Cnn2-targeted mice. Immediately after euthanasia, tissues were rapidly dissected on ice and briefly rinsed in cold phosphate-buffered saline (PBS). Total proteins were extracted from the tissues by mechanical homogenization in SDS-PAGE sample buffer containing 2% SDS and heated at 80 °C for 5 min. SDS-PAGE samples of isolated or cultured cells were prepared similarly, omitting the mechanical homogenization step. The protein extracts were examined by SDS-PAGE using the Laemmli buffer system and Coomassie Blue R-250 staining. Duplicate gels were electrically blotted on nitrocellulose membrane using a Bio-Rad semi-dry transfer apparatus for Western analysis. After blocking with 1% bovine serum albumin or 5% powdered skim milk in Tris-buffered saline, the membrane was incubated with a rabbit antiserum, RAH2, which was raised against mouse h2-calponin immunogen and has a weak cross-reaction to h1-calponin (24Nigam R. Triggle C.R. Jin J.-P. J. Muscle Res. Cell Motil. 1998; 19: 695-703Crossref PubMed Scopus (29) Google Scholar), a mouse anti-h1-calponin monoclonal antibody (mAb) CP1 (25Jin J.-P. Walsh M.P. Resek M.E. McMartin G.A. Biochem. Cell Biol. 1996; 74: 187-196Crossref PubMed Scopus (31) Google Scholar), mouse anti-h2-calponin mAbs CP21 and 1D2 (3Hossain M.M. Crish J.F. Eckert R.L. Lin J.J.-C. Jin J.-P. J. Biol. Chem. 2005; 280: 42442-42453Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar), or anti-tropomyosin mAb LC24 (26Warren K.S. Lin J.L. McDermott J.P. Lin J.J. J. Cell Biol. 1995; 129: 697-708Crossref PubMed Scopus (59) Google Scholar) (provided by Prof. Jim Lin, University of Iowa) in Tris-buffered saline containing 0.1% bovine serum albumin or 0.5% powdered skim milk to examine the expression of calponin and tropomyosin. As described previously (4Hossain M.M. Smith P.G. Wu K. Jin J.-P. Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar), the blots were washed with Tris-buffered saline containing 0.05% Tween 20 and incubated with alkaline phosphatase-labeled anti-rabbit IgG or anti-mouse IgG second antibody (Sigma). After the final washes, the blots were processed for 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chromogenic substrate reaction (4Hossain M.M. Smith P.G. Wu K. Jin J.-P. Biochemistry. 2006; 45: 15670-15683Crossref PubMed Scopus (35) Google Scholar). Densitometry analysis of SDS gels and Western blots were performed on digital images scanned at 600 dots/inch using the NIH Image software version 1.61. Quantification of the SDS gel and Western blots was done by normalization to the amount of actin, total cellular protein, or histones determined in parallel SDS gels. InVitro Differentiation of Human Peripheral Monocytes into Macrophages—Human monocytes were isolated by elutriation from peripheral blood of anonymous healthy donors to start primary cell cultures. This investigation was determined to be exempted research by the Northwestern University Institutional Review Board. The purified peripheral monocytes were suspended in serum-free RPMI 1640 medium, and allowed to adhere to cell culture dishes at 37 °C in 5% CO2 for 1 h before change to medium containing 20% fetal bovine serum (FBS), 100 μg/ml penicillin, 50 μg/ml streptomycin, and 1 μg/ml of polymyxin B. The adherent cells were allowed to differentiate into macrophages in culture for up to 7 days as described previously (27Huang Q.-Q. Ma Y.Y. Adebayo A. Pope R.M. Arthritis Rheum. 2007; 56: 2192-2201Crossref PubMed Scopus (156) Google Scholar, 28Liu H. Perlman H. Pagliari L.J. Pope R.M. J. Exp. Med. 2001; 194: 113-126Crossref PubMed Scopus (191) Google Scholar, 29Liu H. Ma Y. Cole S.M. Zander C. Chen K.H. Karras J. Pope R.M. Blood. 2003; 102: 344-352Crossref PubMed Scopus (129) Google Scholar, 30Liu H. Eksarko P. Temkin V. Haines 3rd, G.K. Perlman H. Koch A.E. Thimmapaya B. Pope R.M. J. Immunol. 2005; 175: 8337-8345Crossref PubMed Scopus (76) Google Scholar, 31Ma Y. Liu H. Tu-Rapp H. Thiesen H.J. Ibrahim S.M. Cole S.M. Pope R.M. Nat. Immunol. 2004; 5: 380-387Crossref PubMed Scopus (118) Google Scholar, 32Pagliari L.J. Perlman H. Liu H. Pope R.M. Mol. Cell Biol. 2000; 20: 8855-8865Crossref PubMed Scopus (141) Google Scholar, 33Perlman H. Pagliari L.J. Georganas C. Mano T. Walsh K. Pope R.M. J. Exp. Med. 1999; 190: 1679-1688Crossref PubMed Scopus (219) Google Scholar). Collection and Immunophenotyping of Mouse Peritoneal Cells—Peritoneal residential cells were lavaged with PBS from wild type and h2-calponin knock-out mice. Mouse peritoneal cells were also elicited by injection of 2 ml of 3% thioglycollate broth 12 and 72 h prior to lavage. A fixed volume (8 ml) of PBS was used for each animal so the total number of cells lavaged could be compared. Cell types are identified by immunophenotyping with florescence-conjugated antibodies recognizing specific cell surface markers of myeloid cell (Mac-1), macrophage (F4/80), and granulocyte (Gr-1) followed by flow cytometry analysis. 5 × 105 cells from each mouse were first incubated with anti-mouse CD16/CD32 antibody (BD Biosciences) to block the cell surface Fc III/II receptor and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-Mac1 (Mac1-FITC, Serotec), allophycocyanin-conjugated anti-F4/80 (F4/80-allophycocyanin, Serotec), and phycoerythrin-Cy7 (PEcy7)-conjugated anti-Gr1 (Gr1-PEcy7, eBioscience, CA) antibodies at room temperature for 30 min. After washing away the unbound antibodies, the cells were analyzed using a BD LSR II flow cytometer (BD Biosciences) with BD FACSDIVA software. Using the FCS Express software (DeNovo Software, Los Angeles, CA), macrophages were identified as strong Mac1-positive, F4/80-positive, and Gr1-negative, whereas granulocytes were identified as strong Mac1-positive, F4/80-negative, and Gr1-positive. In Vitro Wound Healing Assay—Residential mouse peritoneal cells were collected in RPMI 1640 medium containing 10% FBS, 100 μg/ml penicillin, and 50 μg/ml streptomycin and seeded in 12-well culture plates at 1 × 106 per well to allow high density adhesion of macrophages. After removing the floating cells, the adherent cells were incubated at 37 °C in 5% CO2 for 24 h to form a confluent monolayer. The macrophage monolayer was wounded by scratching with a thin pipette tip and examined at a series of time points under a phase-contrast microscope. The width of the wounds was measured from photographs to evaluate the rate of cell migration during the course of healing. In Vitro Proliferation of Mouse Bone Marrow Cells—Bone marrow cells were flushed out from femora and tibias of 3-month-old wild type and h2-calponin knock-out mice. The cells were induced to differentiate into macrophages in DMEM containing 10% FBS, 20% L929 cell-conditioned media (34Davies J.Q. Gordon S. Helgason C.D. Miller C.L. Basic Cell Culture Protocols. Third Edition. 290. Humana Press Inc., Totowa, NJ2005: 91-103Google Scholar), 5% horse serum, 1% sodium pyruvate, 2 mm l-glutamine, 100 μg/ml penicillin, and 50 μg/ml streptomycin at 37 °C in 5% CO2. After 4 days of culture, the adherent cells were collected with a cell scraper and re-seeded in 96-well plates at 2.5 × 103 cells per well in 200 μl of the same media. The rate of cell proliferation in culture was examined in quadruplet wells at a series of time points by Crystal Violet staining of nuclei as described previously (18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). Transfection of RAW Cells—RAW264.7 mouse macrophage cells (35Raschke W.C. Baird S. Ralph P. Nakoinz I. Cell. 1978; 15: 261-267Abstract Full Text PDF PubMed Scopus (639) Google Scholar) (ATCC TIB-71) were seeded at 2 × 106 cells/100-mm dish in DMEM containing 10% FBS, 100 μg/ml penicillin, and 50 μg/ml streptomycin at 37 °C in 5% CO2 and cultured for 24 h prior to transfection with recombinant pcDNA3.1 plasmid DNA expressing mouse h2-calponin under the cytomegalovirus promoter (18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). Two μg of DNA in 50 μl of DMEM was mixed with 5 μl of Lipofectamine (Invitrogen) and incubated at room temperature for 20 min before being gently mixed with 5 ml of DMEM and added to the RAW264.7 cell culture. After incubation at 37 °C in 5% CO2 for 6 h, 5 ml of DMEM containing 20% FBS was added to the dish and the culture was further incubated for 18 h before change to fresh media. To establish RAW264.7 cell lines stably expressing h2-calponin, the transfected cells were selected by G418 (300 μg/ml) and drug-resistant single colonies were individually picked as described previously (18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). The integration of the sense and antisense h2-calponin cDNA transgenes in the stably transfected RAW cell lines was verified by PCR. The expression of h2-calponin in the sense cDNA-transfected cells was examined by Western blotting using the RAH2 antibody. RAW264.7 cells stably transfected with sense and antisense h2-calponin cDNA and non-transfected cells were seeded in 96-well culture plates at 1 × 103 cells per well for cell proliferation assay as described above. Multiple stable transfected cell lines were examined to avoid line to line differences. Immunofluorescence Microscopy—To examine the cellular localization of h2-calponin in macrophages and the effect of Cnn2 knock-out on the structure of macrophage actin cytoskeleton, residential mouse peritoneal macrophages were cultured as a monolayer on glass coverslips for 24 h and stained with anti-h2-calponin antibody RAH2 and normal rabbit serum control, followed by TRITC- or FITC-labeled anti-rabbit IgG second antibody (Sigma) as described previously (18Hossain M.M. Hwang D.-Y. Huang Q.-Q. Sasaki Y. Jin J.-P. Am. J. Physiol. 2003; 284: C156-C167Crossref PubMed Scopus (66) Google Scholar). Actin filaments were stained with TRITC-phalloidin. Tropomyosin was stained with mAb CG3 (36Lin J.J.-C. Chou C.-S. Lin J.L.-C. 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After opsonization by incubation in 50% pooled normal mouse serum diluted in Krebs-Ringer PBS (pH 7.4) for 30 min, red fluorescence carboxyl microspheres (1.0 μm diameter, excitation/emission maxima at 580 nm/605 nm, Molecular Probes) were incubated with mouse residential and elicited peritoneal cells (1 × 106) at a particle to cell ratio of 50:1 in 0.5 ml of RPMI 1640 medium containing a final concentration of 5% mouse serum at 37 °C in 5% CO2 for 1 h. The cells were then thoroughly washed with PBS four times to remove the free and extracellularly bound particles and immediately stained with F4/80-allophycocyanin and Gr1-PEcy7 antibodies as above. After washing to remove the unbound antibodies, the cells were fixed with 1% formalin in PBS. Uptake of the fluorescent beads was measured using a BD FACSArray flow cytometer at 532 nm laser excitation. Phagocytotic activity was analyzed by the FCS Express software. Data Analysis—All quantitative data are presented as mean ± S.D. or S.E. as noted in the figure legends. Statistical analysis was done with Student's t test using Microsoft Excel (two-tail assays unless noted in the figure legends). Significant Levels of H2-calponin in Myeloid Cells—We found that h2-calponin is highly expressed in human peripheral blood mononuclear cells, human myelogenous leukemia cell line K562 (ATCC CCL-243), and human monocyte line THP-1 (ATCC TIB-202) (Fig. 1A). Although h2-calponin is minimally detectable in undifferentiated human promyeloblast line HL-60 (ATCC CCL-240), the expression is significantly up-regulated 5 days after dimethyl sulfoxide-induced differentiation in culture (41Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2458-2462Crossref PubMed Scopus (1427) Google Scholar). 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