Serum response factor regulates expression of phosphatase and tensin homolog through a microRNA network in vascular smooth muscle cells.

OBJECTIVE: Serum response factor (SRF) is a critical transcription factor in smooth muscle cells (SMCs) controlling differentiation and proliferation. Our previous work demonstrated that depleting SRF in cultured SMCs decreased expression of SMC markers but increased proliferation and inflammatory mediators. A similar phenotype has been observed in SMCs silenced for phosphatase and tensin homolog (PTEN), suggesting that SRF and PTEN may lie on a common pathway. Our goal was to determine the effect of SRF depletion on PTEN levels and define mechanisms mediating this effect. METHODS AND RESULTS: In SRF-silenced SMCs, PTEN protein levels but not mRNA levels were decreased, suggesting posttranscriptional regulation. Reintroduction of PTEN into SRF-depleted SMCs reversed increases in proliferation and cytokine/chemokine production but had no effect on SMC marker expression. SRF-depleted cells showed decreased levels of microRNA (miR)-143 and increased miR-21, which was sufficient to suppress PTEN. Increased miR-21 expression was dependent on induction of Fos related antigen (FRA)-1, which is a direct target of miR-143. Introducing miR-143 into SRF-depleted SMCs reduced FRA-1 expression and miR-21 levels and restored PTEN expression. CONCLUSIONS: SRF regulates PTEN expression in SMCs through a miR network involving miR-143, targeting FRA-1, which regulates miR-21. Cross-talk between SRF and PTEN likely represents a critical axis in phenotypic remodeling of SMCs.

Altered expression of genes profiles modulated by a combination of Astragali Radix and Angelicae Sinensis Radix in obstructed rat kidney.

The decoction of a combination of two Chinese herbs, Astragali Radix (the roots of Astragalus membranaceus var. mongholicus) and Angelicae Sinensis Radix (the roots of Angelica sinensis), here named as A&A, has been demonstrated to have renoprotective effects in several animal models and may be considered as a complementary therapeutic medicine for chronic kidney disease. In this study, genomic approaches were employed to identify expression signatures in the obstructed kidney, which may be linked to the molecular actions associated with anti-fibrotic effects of A&A. Ninety-six male Wistar rats were divided randomly into sham, SAA (sham + A&A), UUO (unilateral ureteral obstruction), and UAA (UUO + A&A) groups. The rats in the SAA and UAA groups were administered A&A (14 g/kg) by oral gavage once daily; the ones in the sham and UUO groups were given equal volumes of water. Eight rats from each group were sacrificed at days 3, 7, and 10 after the operation, respectively. Changes in gene expression in the kidneys were determined using Affymetrix RAE-230A GeneChips. The differential expression of known genes between UAA and UUO was confirmed by RT-PCR. The results revealed that 40, 65, and 104 genes were upregulated and 30, 36, and 40 genes downregulated in UUO compared with the sham group at days 3, 7, and 10, respectively. Compared to the UUO group, eight genes were upregulated and two genes were downregulated at day 3 in the UAA group, and two genes were upregulated at day 10. These genes included transient receptor protein 3 (TRP3), bone marrow stromal cell antigen 1 (BST-1), peroxisomal biogenesis factor 6 (PEX6), xanthine dehydrogenase (XDH), cytochrome P450 subfamily I member A1 (CYP1A1), serine/cysteine proteinase inhibitor clade E member1 (PAI-1), fibroblast growth factor 23 (FGF23), and five ESTs. Among these genes, differential expression of PAI-1, FGF23, and CYP1A1 were further confirmed by RT-PCR. These data provide the evidence that the anti-fibrotic effects of A&A are mediated through multiple pathways in obstructive nephropathy, and novel mechanisms may be involved in the increasing degeneration of ECM, decreasing ROS reaction, and regulation of the calcium-phosphate metabolism.

Antitumorigenic effects of peroxisome proliferator-activated receptor-gamma in non-small-cell lung cancer cells are mediated by suppression of cyclooxygenase-2 via inhibition of nuclear factor-kappaB.

Pharmacological activators of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibit growth of non-small-cell lung cancer (NSCLC) cell lines in vitro and in xenograft models. Because these agents engage off-target pathways, we have assessed the effects of PPARgamma by overexpressing the protein in NSCLC cells. We reported previously that increased PPARgamma inhibits transformed growth and invasiveness and promotes epithelial differentiation in a panel of NSCLC expressing oncogenic K-Ras. These cells express high levels of cyclooxygenase-2 (COX-2) and produce high levels of prostaglandin E(2) (PGE(2)). The goal of these studies was to identify the molecular mechanisms whereby PPARgamma inhibits tumorigenesis. Increased PPARgamma inhibited expression of COX-2 protein and promoter activity, resulting in decreased PGE(2) production. Suppression of COX-2 was mediated through increased activity of the tumor suppressor phosphatase and tensin homolog, leading to decreased levels of phospho-Akt and inhibition of nuclear factor-kappaB activity. Pharmacological inhibition of PGE(2) production mimicked the effects of PPARgamma on epithelial differentiation in three-dimensional culture, and exogenous PGE(2) reversed the effects of increased PPARgamma activity. Transgenic mice overexpressing PPARgamma under the control of the surfactant protein C promoter had reduced expression of COX-2 in type II cells and were protected against developing lung tumors in a chemical carcinogenesis model. These data indicate that high levels of PGE(2) as a result of elevated COX-2 expression are critical for promoting lung tumorigenesis and that the antitumorigenic effects of PPARgamma are mediated in part through blocking this pathway.

Depletion of serum response factor by RNA interference mimics the mitogenic effects of platelet derived growth factor-BB in vascular smooth muscle cells.

Promoters of many smooth muscle-specific genes (SM-genes) contain multiple CArG boxes, which represent a binding site for serum response factor (SRF). Transcriptional control through these regions involves interactions with SRF and specific coactivators such as myocardin. We have previously reported that suppression of SM-gene expression by platelet derived growth factor (PDGF) is associated with redistribution of SRF, leading to lower intra-nuclear levels, and a reduction in SRF transactivation. To further assess the role of SRF depletion on VSMC phenotype, the current study used RNA interference (RNAi). Two SRF-specific sequences constructed as hairpins were stably expressed in rat VSMC. Clones expressing SRF RNAi had no detectable SRF expression by immunoblotting, and showed diminished levels of SM alpha-actin protein and promoter activity. Unexpectedly, depletion of VSMC resulted in increased rates of proliferation and migration. Several genes whose expression is increased by PDGF stimulation, including c-Jun, were similarly induced in cells lacking SRF. Effects of SRF depletion were not attributable to altered PDGF receptor activity or alterations in activation of Akt. These data indicate that loss of SRF transactivation in VSMC, in this case through suppression via RNAi, induces biological responses similar to that seen with PDGF.

Peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) inhibits tumorigenesis by reversing the undifferentiated phenotype of metastatic non-small-cell lung cancer cells (NSCLC).

Pharmacological activators of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) have been shown to inhibit growth of lung tumors largely through growth inhibition and induction of apopotosis. However, since many of these agents engage other effectors, the role of (PPAR(gamma) in lung tumorigenesis remains poorly defined. To specifically examine PPAR(gamma)-mediated events, non-small-cell lung cancer (NSCLC) cells overexpressing PPAR(gamma) were established. Overexpression of PPAR(gamma) in H2122 adenocarcinoma cells (H2122-PPAR(gamma)) blocked anchorage-independent growth compared to cells transfected with empty vector (H2122-LNCX), but had no significant effect on cell proliferation or apoptosis under standard tissue culture conditions. Orthotopic implantation of H2122-PPAR(gamma) cells into the lungs of nude rats inhibited tumor growth and metastasis in vivo and prolonged survival compared to implantation of H2122-LNCX cells. Consistent with these findings, H2122-PPAR(gamma) cells had an impaired invasiveness as assessed in Transwell assays. In a three-dimensional culture system, H2122-PPAR(gamma) cells formed polarized spheroid structures similar to those observed with normal lung epithelial cells. H2122-LNCX cells formed nonpolarized aggregate structures and did not show any of these epithelial properties. These data indicate that inhibitory effects of PPAR(gamma) on lung tumorigenesis involve selective inhibition of invasive metastasis, and activation of pathways that promote a more differentiated epithelial phenotype.

Lung Krüppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A2 alpha promoter.

Increased expression of cPLA2 (cytosolic phospholipase A2) has been shown to be the cause of tumorigenesis of NSCLC (non-small-cell lung cancer). Our laboratory has previously demonstrated that oncogenic forms of Ras increase transcription of cPLA2 in normal lung epithelial cells and NSCLC lines through activation of the ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) MAPK (mitogen-activated protein kinase) family. We have also defined a minimal region of the cPLA2 promoter that is critical for this induction. To identify potential transcription factors that bind to this region and regulate expression, a yeast one-hybrid screen was performed with a rat lung cDNA library. Multiple members of the Krüppel family were identified, with LKLF (lung Krüppel-like factor) being isolated a number of times. Overexpression of LKLF in lung epithelial cells or Drosophila SL-2 cells increased cPLA2 promoter activity. Conversely, expression of a dominant negative form of LKLF inhibited induction of cPLA2 promoter activity by oncogenic Ras in normal lung epithelial cells and NSCLC. By electrophoretic mobility-shift assay analysis, it was found that LKLF bound to a GC-rich region of the cPLA2 promoter located between -37 and -30 upstream from the transcription start site. Expression of siRNA (small interfering RNA) directed against LKLF inhibited basal expression of cPLA2 in lung epithelial cells and blocked induction by H-Ras. In NSCLC, siRNA against LKLF co-operated with siRNA against Sp1 (stimulatory protein 1) to inhibit cPLA2 promoter activity. Finally, recombinant LKLF was a substrate for ERKs. These results indicate that LKLF is an important regulator of cPLA2 expression and participates in the induction of this protein, which is critical for increased eicosanoid production associated with lung tumorigenesis.

Oncogenic K-Ras regulates proliferation and cell junctions in lung epithelial cells through induction of cyclooxygenase-2 and activation of metalloproteinase-9.

Expression of oncogenic K-Ras is frequently observed in non-small-cell lung cancer. However, oncogenic K-Ras is not sufficient to transform lung epithelial cells and requires collaborating signals that have not been defined. To examine the biological effects of K-Ras in nontransformed lung epithelial cells, stable transfectants were generated in RL-65 cells, a spontaneously immortalized lung epithelial cell line. Expression of K-Ras resulted in extracellular signal-regulated kinase (ERK) activation, which mediated induction of cyclooxygenase (COX)-2 and increased prostaglandin E(2) production. Epithelial cells expressing oncogenic K-Ras showed increased proliferation in two- and three-dimensional tissue culture and delayed formation of hollow acinar structures in three-dimensional matrigel cultures. These affects were mediated through COX-2-dependent activation of beta-catenin signaling and inhibition of apoptosis. ERK activation also led to induction of metalloproteinase (MMP)-9 and cleavage of E-cadherin at two specific sites. This resulted in partial disruption of adherens junctions as determined by decreased transepithelial resistance (TER), and disruption of E-cadherin/beta-catenin interactions. An MMP-9 inhibitor reversed the decrease in TER and inhibited beta-catenin signaling. These data indicate that although expression of oncogenic K-Ras does not transform lung epithelial cells, it alters the phenotype of the cells by increasing proliferation and decreasing cell-cell contacts characteristic of epithelial cells.

Depletion of cytosolic phospholipase A2 in bone marrow-derived macrophages protects against lung cancer progression and metastasis.

Cancer progression and metastasis involves interactions between tumor cells and the tumor microenvironment (TME). We reported that mice deficient for cytosolic phospholipase A(2) (cPLA(2)-KO) are protected against the development of lung tumors. The goal of this study was to examine the role of cPLA(2) in the TME. Mouse lung cancer cells (CMT167 and Lewis lung carcinoma cells) injected directly into lungs of syngeneic mice formed a primary tumor, and then metastasized to other lobes of the lung and to the mediastinal lymph nodes. Identical cells injected into cPLA(2)-KO mice showed a dramatic decrease in the numbers of secondary metastatic tumors. This was associated with decreased macrophage staining surrounding the tumor. Wild-type mice transplanted with cPLA(2)-KO bone marrow had a marked survival advantage after inoculation with tumor cells compared with mice receiving wild-type (WT) bone marrow. In vitro, coculturing CMT167 cells with bone marrow-derived macrophages from WT mice increased production of interleukin 6 (IL-6) by cancer cells. This increase was blocked in cocultures using cPLA(2)-KO macrophages. Correspondingly, IL-6 staining was decreased in tumors grown in cPLA(2)-KO mice. These data suggest that stromal cPLA(2) plays a critical role in tumor progression by altering tumor-macrophage interactions and cytokine production.

Patterns of gene expression differentially regulated by platelet-derived growth factor and hypertrophic stimuli in vascular smooth muscle cells: markers for phenotypic modulation and response to injury.

In vascular smooth muscle cells (VSMC), platelet-derived growth factor (PDGF) suppresses expression of multiple smooth muscle contractile proteins, useful markers of differentiation. Conversely, hypertrophic agents induce expression of these genes. The goal of this study was to employ genomic approaches to identify classes of genes differentially regulated by PDGF and hypertrophic stimuli. Changes in gene expression were determined using Affymetrix RAE-230 GeneChips in rat aortic VSMC stimulated with PDGF. For comparison with a model hypertrophic stimulus, a microarray was performed with VSMC stably expressing constitutively active Galpha(16), which strongly induces smooth muscle marker expression. We identified 75 genes whose expression was increased by exposure to PDGF and decreased by expression of Galpha(16) and 97 genes whose expression was decreased by PDGF and increased by Galpha(16). These genes included many smooth muscle-specific proteins; several extracellular matrix, cytoskeletal, and chemotaxis-related proteins; cell signaling molecules; and transcription factors. Changes in gene expression for many of these were confirmed by PCR or immunoblotting. The contribution of signaling pathways activated by PDGF to the gene expression profile was examined in VSMC stably expressing gain-of-function H-Ras or myristoylated Akt. Among the genes that were confirmed to be differentially regulated were CCAAT/enhancer-binding protein delta, versican, and nexilin. All of these genes also had altered expression in injured aortas, consistent with a role for PDGF in the response of injured VSMC. These data indicate that genes that are differentially regulated by PDGF and hypertrophic stimuli may represent families of genes and potentially be biomarkers for vascular injury.

Induction of SM-alpha-actin expression by mechanical strain in adult vascular smooth muscle cells is mediated through activation of JNK and p38 MAP kinase.

Mechanical forces have direct effects on the growth and differentiation of vascular smooth muscle. The goal of this study was to examine the effects of cyclic mechanical strain on expression of smooth muscle-alpha-actin (SM-alpha-actin), a marker for the differentiated state of vascular smooth muscle, in cultured rat aortic smooth muscle cells (VSMC). Cells grown on dishes coated with either laminin or pronectin were subjected to mechanical strain and effects on expression of SM-alpha-actin were evaluated using the Flexercell Strain Unit. Application of mechanical strain to cells in full media increased SM-alpha-actin protein expression and promoter activity. This was not associated with any effect on growth. Mechanical strain increased activity of all three members of the MAP kinase family (ERKs, JNKs, and p38 MAP kinase), with similar kinetics. Inhibition of either JNKs or p38 MAP kinase blocked the strain-induced increase in SM-alpha-actin promoter activity, and expression of constitutively active forms of JNK or MKK6, a p38 kinase, increased promoter activity. These studies indicate that in adult VSMC, mechanical strain leads to increased expression of smooth muscle markers, resulting in a more contractile phenotype.

Regulation of SM22 alpha expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells.

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM alpha-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22 alpha appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22 alpha expression by AVP and PDGF in cultured VSMC. Levels of SM22 alpha mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22 alpha promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22 alpha promoter. Expression of constitutively active Ras produced similar suppressions on SM22 alpha promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.

Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells.

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.