HLA-G is a nonclassic, class I HLA molecule that has important immunomodulatory properties. Previously, we identified HLA-G as an asthma-susceptibility gene and discovered that the risk of asthma in a child was determined by both the child's HLA-G genotype and the mother's affection status. Here we report a SNP in the 3' untranslated region of HLA-G that influences the targeting of three microRNAs (miRNAs) to this gene, and we suggest that allele-specific targeting of these miRNAs accounts, at least in part, for our earlier observations on HLA-G and the risk of asthma.

We tested the hypothesis that prolonged serum deprivation would allow a subset of cultured airway myocytes to reacquire the abundant contractile protein content, marked shortening capacity, and elongated morphology characteristic of contractile cells within intact tissue. Passage 1 or 2 canine tracheal smooth muscle (SM) cells were grown to confluence, then serum deprived for up to 19 days. During serum deprivation, two differentiation pathways emerged. One-sixth of the cells developed an elongated morphology and aligned into bundles. Elongated myocytes contained cables of contractile myofilaments, dense bodies, gap junctions, and membrane caveoli, ultrastructural features of contractile SM in tissue. These cells immunostained intensely for SM α-actin, SM myosin heavy chain (MHC), and SM22 (an SM-specific actin-binding protein), and Western analysis of culture lysates disclosed 1.8 (SM α-actin)-, 7.7 (SM MHC)-, and 5.8 (SM22)-fold protein increases during serum deprivation. Immunoreactive M3 muscarinic receptors were present in dense foci distributed throughout elongated, SM MHC-positive myocytes. ACh (10−3 M) induced a marked shortening (59.7 ± 14.4% of original length) in 62% of elongated myocytes made semiadherent by gentle proteolytic digestion, and membrane bleb formation (a consequence of contraction) occurred in all stimulated cells that remained adherent and so did not shorten. Cultured airway myocytes that did not elongate during serum deprivation instead became short and flattened, lost immunoreactivity for contractile proteins, lacked the M3 muscarinic-receptor expression pattern seen in elongated cells, and exhibited no contractile response to ACh. Thus we demonstrate that prolonged serum deprivation induces distinct differentiation pathways in confluent cultured tracheal myocytes and that one subpopulation acquires an unequivocally functional contractile phenotype in which structure and function resemble contractile myocytes from intact tissue.

SM22 is a 201-amino acid actin-binding protein expressed at high levels in smooth muscle cells. It has structural homology to calponin, but how SM22 binds to actin remains unknown. We performed site-directed mutagenesis to generate a series of NH(2)-terminal histidine (His)-tagged mutants of human SM22 in Escherichia coli and used these to analyze the functional importance of potential actin binding domains. Purified full-length recombinant SM22 bound to actin in vitro, as demonstrated by cosedimentation assay. Binding did not vary with calcium concentration. The COOH-terminal domain of SM22 is required for actin affinity, because COOH terminally truncated mutants [SM22-(1-186) and SM22-(1-166)] exhibited markedly reduced cosedimentation with actin, and no actin binding of SM22-(1-151) could be detected. Internal deletion of a putative actin binding site (154-KKAQEHKR-161) partially prevented actin binding, as did point mutation to neutralize either or both pairs of positively charged residues at the ends of this region (KK154LL and/or KR160LL). Internal deletion of amino acids 170-180 or 170-186 also partially or almost completely inhibited actin cosedimentation, respectively. Of the three consensus protein kinase C or casein kinase II phosphorylation sites in SM22, only Ser-181 was readily phosphorylated by protein kinase C in vitro, and such phosphorylation greatly decreased actin binding. Substitution of Ser-181 to aspartic acid (to mimic serine phosphorylation) also reduced actin binding. Immunostains of transiently transfected airway myocytes revealed that full-length NH(2)-terminal FLAG-tagged SM22 colocalizes with actin filaments, whereas FLAG-SM22-(1-151) does not. These data confirm that SM22 binds to actin in vitro and in vivo and, for the first time, demonstrate that multiple regions within the COOH-terminal domain are required for full actin affinity.

Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells.

RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics.

In the sentence ''Thus, consistent with our earlier report in two other Caucasian populations, the þ3142G allele, or promoter alleles in strong LD with þ3142G (r 2 ¼ 0.76 between þ3142 and either À964 or À1306), is a susceptibility allele among children of asthmatic mothers, and the C allele may be a risk allele (albeit more modest) among children of non-asthmatic mothers,'' the G and C alleles should be switched. The authors regret the error.

MicroRNA (miR)-146a and miR-146b are negative regulators of inflammatory gene expression in lung fibroblasts, epithelial cells, monocytes, and endothelial cells. The abundance of cyclooxygenase-2 (COX-2) and IL-1β is negatively regulated by the miR-146 family, suggesting miR-146a and/or miR-146b might modulate inflammatory mediator expression in airway smooth muscle thereby contributing to pathogenesis of asthma. To test this idea we compared miR-146a and miR-146b expression in human airway smooth muscle cells (hASMCs) from nonasthmatic and asthmatic subjects treated with cytomix (IL-1β, TNF-α, and IFNγ) and examined the miRNAs' effects on COX-2 and IL-1β expression. We found that cytomix treatment elevated miR-146a and miR-146b abundance. Induction with cytomix was greater than induction with individual cytokines, and asthmatic cells exhibited higher levels of miR-146a expression following cytomix treatment than nonasthmatic cells. Transfection of miR-146a or miR-146b mimics reduced COX-2 and IL-1β expression. A miR-146a inhibitor increased COX-2 and IL-1β expression, but a miR-146b inhibitor was ineffective. Repression of COX-2 and IL-1β expression by miR-146a correlated with reduced abundance of the RNA-binding protein human antigen R. These results demonstrate that miR-146a and miR-146b expression is inducible in hASMCs by proinflammatory cytokines and that miR-146a expression is greater in asthmatic cells. Both miR-146a and miR-146b can negatively regulate COX-2 and IL-1β expression at pharmacological levels, but loss-of-function studies showed that only miR-146a is an endogenous negative regulator in hASMCs. The results suggest miR-146 mimics may be an attractive candidate for further preclinical studies as an anti-inflammatory treatment of asthma.

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcriptionpromoting activity.Confluent cultured, passaged canine tracheal myocytes exhibit divergent phenotypes when deprived of serum for 7 or more days. About 1/6 of these cells accumulate abundant contractile apparatus proteins, increasing whole culture contents of smooth muscle myosin heavy chain (smMHC) 1 and SM22 by 5-7-fold (1, 2). These myocytes acquire a contractile phenotype, characterized by morphological elongation, expression of functionally coupled muscarinic M 3 surface receptors, and substantial contraction (shortening) upon cholinergic stimulation. Presently, the mechanism responsible for this phenotypic differentiation is unknown.In cultured skeletal muscle, differentiation of myoblasts into myotubes depends upon up-regulation of skeletal muscle-specific gene transcription, which results in abundant contractile apparatus protein accumulation (3-6). This precedent in skeletal muscle suggested that similarly enhanced transcription of contractile apparatus genes might account for the substantial smMHC and SM22 accumulation we observed in long term serum-deprived tracheal smooth muscle cells. To test this hypothesis, we assessed transcription from the smMHC and SM22 gene promoters in both preconfluent serum fed and post confluent long term serum-deprived tracheal myocytes. Contrary to our expectation, we found markedly reduced transcription from...