PAC1 Cells Research Articles

RAB5A promotes the formation of filopodia in pancreatic cancer cells via the activation of cdc42 and β1-integrin

Filopodia are slender actin-rich plasma membrane protrusions that function to drive cell migration and invasion. Despite the observation of defective filopodia formation in many malignant tumors, the regulation mechanism remained unknown to date. In the present study, for the first time, we demonstrate that RAB5A, a Rab GTPase family protein, is a potent regulator of filopodia formation in pancreatic cancer cells. High expression of RAB5A was associated with filopodia formation and migration in pancreatic cancer cells. Overexpression of RAB5A promoted filopodia formation and migration in CF Pac-1 cells. In contrast, down-regulation of RAB5A expression in SW1990 cells with a high endogenous RAB5A expression level impeded the formation of filopodia. Further analysis indicated that RAB5A was required for cdc42 activation in CF Pac-1 and SW1990 cells. Moreover, to investigate the underlying mechanism by which the activation of cdc42 mediates RAB5A-induced filopodia formation, the active state of β1-integrin was examined in cells with different expression levels of RAB5A. We observed that RAB5A regulated the accumulation of the active β1-integrin. We demonstrated that down-regulation of the expression of β1-integrin strongly suppressed filopodia formation and cdc42 activation mediated by RAB5A. These results indicate the important role of RAB5A in the regulation of filopodia formation in pancreatic cancer cells, which is dependent on the activation of cdc42 and β1-integrin.

Fli1 Expression Level Affects Megakaryopoiesis and Thrombopoiesis: More Is Not Always a Bad Thing

Friend leukemia integration 1 (FLI1) is a critical transcription factor responsible for terminal megakaryocyte differentiation. This transcription factor is amongst the genes missing in the inherited disorder Jacobsen syndrome, resulting from a hemizygous deletion on chromosome 11q. The deletion causes dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Also, FLI1 mutations in its DNA-binding domain region results in thrombocytopenia in affected patients. We examined induced pluripotent stem cell- (iPSC) derived megakaryocytes (iMegs) to determine if the platelet disorder observed in PTSx could be replicated and if varied levels of Fli1 expression affected megakaryopoiesis and thrombopoiesis. Beginning with a normal control (WT) iPSC line, genome editing was performed to generate three lines with: 1) one copy of FLI1 disrupted (FLI1+/-), 2) hemizygous transgene expression of Fli1 in the adeno-associated virus site 1 (AAVS1) safe harbor locus using a megakaryocyte-specific GP1balpha promoter (WT-overexpressing line, WT-OE), and 3) homozygous transgene expression in AAVS1 (WT-OE2). Additionally, we established an iPSC line from a PTSx patient and edited this line for a similar hemizygous transgene expression of FLI1 (PTSx-OE). Data described here are summarized in the tables below. We confirm our genome editing strategies by examining mRNA and protein levels of iMegs and found WT-OE and WT-OE2 iMegs have ~3X higher mRNA and ~7X higher protein levels than WT iMegs. FLI1+/- and PTSx iMegs both have lower mRNA and ~0.5X the protein levels of WT iMegs, while PTSx-OE iMegs had comparable levels of mRNA and protein as WT-OE iMegs. Megacult colony assays showed WT-OE and WT-OE2 yielded more CFU-Megs than WT HPCs (p²0.01, p²0.05, respectively). PTSx and FLI1+/- had much less CFU-Megs compared to PTSx-OE and WT (p²0.0001). After growth in liquid culture, WT-OE and -OE2 lines had an increase in iMeg numbers (p=0.29, p²0.01) while FLI1+/- and PTSx iMeg numbers were 43% and 10% that of the WT control (p²0.01, p²0.001). PTSx-OE iMeg numbers were comparable to WT. Surface marker CD41 and CD42b levels were increased compared to WT iMegs in the WT-OE and -OE2 iMegs (p=0.25, p²0.05) and less on FLI1+/- and PTSx iMegs (p²0.01, p²0.05). PTSx-OE iMegs were normal compared to WT, but p²0.05 vs. PTSx. Infused CD41+CD42b+ iMegs into NSG mice showed a trend toward same yield of platelets in the OE lines and lower yield in FLI1+/- and PTSx lines compared to WT. However, when calculations were made from HPCs rather than iMegs infused, the FLI1+/- and PTSx iMegs generated significantly less number of released platelets (p²0.05, p²0.01). The half-life of WT-OE and -OE2 released platelets were increased at 4 and 10 hours compared to WT, whereas FLI1+/- and PTSx released platelets have lower half-lives of 2 hours and 0.5 hours, respectively. These decreased platelet half-lives are due to the majority of platelets being defective and cleared at a higher rate. This was corrected to WT in the PTSx-OE-released platelets. Both in vitro iMegs and in vivo released platelets within the murine blood were assessed for function. Analysis of activation by thrombin was performed via FACS for PAC-1 binding and cell surface P-selectin levels, which are both indicators of platelet activation. Initial studies show no difference between WT and the WT-OE and WT-OE2 lines, but FLI1+/- platelet activation was slightly impaired. In summary, we show that studies of iMegs with decreased Fli1 levels replicate many of the clinical features previously described: less iMegs, lower CD41 and CD42b density with less platelets released while having shorter half-lives. On the other hand, increased Fli1 levels resulted in higher number of iMegs with more surface antigen density and released platelets with increased half-lives. Based on overexpression studies of other transcription factors during megakaryopoiesis, such as GATA1, the expectation would have been that the excess of Fli1 in the OE lines would have also resulted in defects in megakaryopoiesis and thrombopoiesis, but instead improvements were seen. The basis for this difference when overexpressing different transcriptional factors needs further analysis. That high Fli1 levels enhance iMeg yield with maintained numbers and function of released platelets may be of value for generating platelets for clinical use from in vitro grown Megs. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Stimulatory effect of pituitary adenylate cyclase-activating polypeptide 6-38, M65 and vasoactive intestinal polypeptide 6-28 on trigeminal sensory neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts on G protein-coupled receptors: the specific PAC1 and VPAC1/VPAC2 receptors. PACAP6-38 was described as a potent PAC1/VPAC2 antagonist in several models, but recent studies reported its agonistic behaviors proposing novel receptorial mechanisms. Since PACAP in migraine is an important research tool, we investigated the effect of PACAP and its peptide fragments on trigeminal primary sensory neurons.Effect of the peptides was studied with ratiometric Ca-imaging technique using the fluorescent indicator fura-2 AM on primary cultures of rat and mouse trigeminal ganglia (TRGs) neurons. Specificity testing was performed on PAC1, VPAC1 and VPAC2 receptor-expressing cell lines with both fluorescent and radioactive Ca-uptake methods.Slowly increasing intracellular free calcium concentration [Ca2+]i was detected after PACAP1-38, PACAP1-27, vasoactive intestinal polypeptide (VIP) and the selective PAC1 receptor agonist maxadilan administration on TRG neurons, but interestingly, PACAP6-38, VIP6-28 and the PAC1 receptor antagonist M65 also caused similar activation. The VPAC2 receptor agonist BAY 55-9837 induced similar activation, while the VPAC1 receptor agonist Ala11,22,28VIP had no significant effect on [Ca2+]i. It was proven that the Ca2+-influx originated from intracellular stores using radioactive calcium-45 uptake experiment and Ca-free solution. On the specific receptor-expressing cell lines the antagonists inhibited the stimulating actions of the respective agonists, but had no effects by themselves.PACAP6-38, M65 and VIP6-28, which were described as antagonists in numerous studies in several model systems, act as agonists on TRG primary sensory neurons. Currently unknown receptors or splice variants linked to distinct signal transduction pathways might explain these differences.

Insulin‐regulated expression of adiponectin receptors in muscle and fat cells

Adp (adiponectin), an adipocyte-secreted hormone, exerts its effect via its specific receptors, AdipoR1 and AdipoR2 (adiponectin receptors 1 and 2), on insulin-sensitive cells in muscle, liver and adipose tissues, and plays an important role in lipid and glucose metabolisms. The study has investigated the effect of insulin on AdipoRs expression in muscle and fat cells. Differentiated fat [3T3-L1 (mouse adipocytes)], L6 (skeletal muscle) and vascular smooth muscle (PAC1) cells were serum starved and exposed to 100 nM insulin for 1-24 h. AdipoR1 and AdipoR2 mRNAs expression was monitored by real-time PCR. The results demonstrate that insulin down-regulates both AdipoR1 and AdipoR2 mRNAs levels in a biphasic manner in L6 and PAC1 cells. Insulin had little or no effect in the regulation of AdipoR1 expression in 3T3-L1 cells, but significantly up-regulated AdipoR2 mRNA level in a biphasic manner. The fact that insulin differentially regulates the expression of AdipoR1 and AdipoR2 in muscle and fat cells suggests this is also dependent on the availability of the endogenous ligand, such as Adp for AdipoR1 and AdipoR2 in fat cells. The effects of globular Adp were also tested on insulin-regulated expression of AdipoRs in L6 cells, and found to up-regulate and counter insulin-mediated suppression of AdipoRs expression in L6 cells.

Abstract 477: SM22 Deficiency Induces SRF Nuclear Translocation and Promotes Carotid Medial Fibrosis in Response to Injury

Introduction: SM22 (aka transgelin) is known to be an actin-binding protein that is rapidly downregulated in response to vascular injury. Despite prevalent view regarding SM22 as a maker of VSMC dedifferentiation, recently a series of studies including our own demonstrate that SM22 downregulation promotes SMC dedifferentiate into a variety of SMC subtypes involving in proliferation, migration, inflammation, osteochondrogendesis and apoptosis. Hypothesis: In the present study, we assess the hypothesis that SM22 regulates the expression of col1a2, a fibrotic marker, in SMCs. Methods: We use molecular and cellular approaches to analyze the regulation of col1a2 in smooth muscle cells upon artery injury using Sm22 knockout mice. Results: We found that carotid injury induces high expression of Col1a2 in the medial layer of the vessel wall and the nuclear translocation of serum response factor (SRF), a key transcription factor that regulates muscle-specific genes as well as growth factor response genes. To determine whether col1a2, an extracellular matrix protein, is a target of SRF, we performed bioinformatics analysis: we identified an evolutionarily conserved SRF binding sequence, CC(A/T)6GG (known as the CArG box), in the col1a2 promoter. Luciferase transfection assay showed that Vp16-SRF significantly transactivated the col1a2 promoter; mutation of the CArG box abolished most of this stimulating effect. Gel shift and chromatin precipitation assays confirmed that SRF bound to the CArG box of the col1a2 promoter. To investigate whether loss of SM22 in SMCs contributes to increased nuclear SRF expression, we knocked down SM22 in PAC1 cells, a VSMC cell line, using SM22 siRNA. Consistent with the in vivo observation, knockdown SM22 also enhanced nuclear SRF expression. However, cola2 transcription was not upregulated, suggesting that additional signals may be involved in this regulation in vivo . Conclusions: SM22 deficiency increases the fibrosis in the arterial media, and the induction of nuclear SRF may contribute at least in part to col1a2 transcription in response to artery injury.

Abstract 18326: p38MAPK is a Novel Negative Regulator for Smooth Muscle Differentiation

p38MAPK belongs to members of the MAPK family which can be activated by various environmental stressors and inflammatory cytokines. While more and more studies have defined the critical roles of p38MAPK in different biological processes involving cell growth, differentiation and apoptosis, its actual contribution to smooth muscle cell (SMC) phenotypic modulation is largely unexplored. Using the mouse complete carotid artery ligation model, we found both total p38 and phosphorylated p38 to be predominantly located in the neointima of the vessel wall where SMCs undergo hyper-proliferation and SMC differentiation is compromised, suggesting a negative regulatory role of p38MAPK in SMC differentiation. To test this hypothesis, we applied siRNA studies in primary human coronary artery SMC (HCASM) cultures and examined SMC contractile gene expression. Depletion of p38MAPK evoked a dramatic increase in SMC contractile gene expression. The stimulatory effect of SMC contractile gene expression upon knockdown of p38MAPK was reproduced in a variety of SMC culture models including human aortic SMCs, rat aortic SMCs, PAC1 cells and primary human myofibroblasts. Consistent with the siRNA studies, cotransfecting p38 with its upstream kinase MKK3 or MMK6 abolished promoter activity of several SMC-restricted genes, while little effect was found with the dominant negative mutants of p38. Furthermore, adenovirus carrying the constitutively active MKK6 completely abrogated TGF-beta1 induced SMC differentiation as assessed by Western blotting and immunfluorescence mirocsopy, indicating that phosphorylated p38 is required for the repressive effect of p38MAPK in SMC differentiation. Interestingly, in HCASMs, we found myocardin-related transcription factor A (MRTFA) was mainly located in the nucleus when the endogenous p38MAPK was knocked down; conversely, over-expression of constitutively active MKK6 was found to shift the majority of MRTFA to the cytoplasm of HCASMs, suggesting that phosphorylated p38MAPK is able to block nuclear import of MRTFA in SMCs. Taken together, our studies revealed an unexpected role for p38MAPK in the negative regulation of SMC differentiation.

Collagen Triple Helix Repeat Containing-1 Induced Upregulation of Myogenin and Decorin Expression

Transforming growth factor-b (TFG-b) and Wnt signaling are implicated in fibrotic processes. Collagen Triple Helix Repeat Containing-1 (Cthrc1) has been shown to increase cell migration, inhibit TGF- b and canonical Wnt signaling, while amplifying noncanonical Wnt signaling. TGF-b has also been show to be a potent inhibitor of myogenesis in cultured myoblasts, due to increased SMAD3 activation and suppressed MyoD and myogenin expression. Decorin is a TGF-b agonist that appears unregulated during skeletal muscle differentiation and is secreted by differentiated myotubes. PURPOSE: To characterize the interactions of Cthrc1, decorin, TGF-b, and Wnt5 in the myogenesis of murine myoblasts. METHODS: C2C12 (mouse skeletal muscle) myoblasts were cultured until 90% confluent in DMEM supplemented with 10% FBS and 1% Pen/Strep. Cells were cultured in triplicate for 48 hrs in the following conditions + or - TGF-b: Control, C2C12 [Cthrc1] (via transfection of adenovirus containing full length Cthrc1), C2C12 [Wnt5a] (conditioned media harvested from L5 cell culture). TGF-b (5ng/ml) was added 30m prior to harvest. Separate cultures were harvested for either RT-PCR or Western blot analyses. RESULTS: Addition of Cthrc1 to C2C12 cells resulted in increased expression of both decorin and myogenin mRNA. The addition of Wnt5a or Cthrc1 was not sufficient to overcome the inhibition of decorin or myogenin expression seen by the addition of TGF-b. Previous work has suggested that both Cthrc1 and Wnt5a may be necessary to overcome TGF-b induced myogenic inhibition. Preliminary western blotting data showed no detectable differences in phospho-p38 levels between control and Cthrc1-conditioned media cultures in C2C12 cell lines. Pac1 cells treated with Cthrc1-conditioned media showed decreased pAKT levels compared to controls and Wnt conditioned cells. AKT activity was also diminished in Pac1 cells transduced with Cthrc1 virus as shown by decreased levels of pGSK3-b. CONCLUSION: Cthrc1 appears to promote skeletal myoblast differentiation, in a TGF-b-regulated manner. Its role in hypertrophy is poorly defined but may involve synergistic signaling with non-canonical Wnts to inhibit TGF-b induced myogenesis.

Disruption of SM22 Promotes Inflammation After Artery Injury via Nuclear Factor κB Activation

SM22 (or transgelin), an actin-binding protein abundant in vascular smooth muscle cells (VSMCs), is downregulated in atherosclerosis, aneurysm and various cancers. Abolishing SM22 in apolipoprotein E knockout mice accelerates atherogenesis. However, it is unclear whether SM22 disruption independently promotes arterial inflammation. To investigate whether SM22 disruption directly promotes inflammation on arterial injury and to characterize the underlying mechanisms. Using carotid denudation as an artery injury model, we showed that Sm22 knockout (Sm22(-/-)) mice developed enhanced inflammatory responses with higher induction of proinflammatory genes, including Vcam1, Icam1, Cx3cl1, Ccl2, and Ptgs2. Higher expression of these genes was confirmed in primary Sm22(-/-) VSMCs and in PAC1 cells after Sm22 knockdown, whereas SM22 recapitulation in primary Sm22(-/-) VSMCs decreased their expression. NFKB2 was prominently activated in both injured carotids of Sm22(-/-) mice and in PAC1 cells after Sm22 knockdown and may mediate upregulation of these proinflammatory genes. As a NF-kappaB activator, reactive oxygen species (ROS) increased in primary Sm22(-/-) VSMCs and in PAC1 cells after Sm22 knockdown. ROS scavengers blocked NF-kappaB activation and induction of proinflammatory genes. Furthermore, Sm22 knockdown increased Sod2 expression and activated p47phox, reflecting contributions of mitochondria and NADPH oxidase to the augmented ROS production; this may result from actin and microtubule cytoskeletal remodeling. Our findings show that SM22 downregulation can induce proinflammatory VSMCs through activation of ROS-mediated NF-kappaB pathways. This study provides initial evidence linking VSMC cytoskeleton remodeling with arterial inflammation.

Abstract 4950: Loss of SM22 Couples Arterial Inflammation With Osteochondrogenesis in a Murine Artery Injury Model

Expression of vascular smooth muscle cell (VSMC) cytoskeleton markers including SM22 is down-regulated in arterial diseases including atherosclerosis where inflammation and calcification are present. However, the role of this down-regulation in arterial pathogenesis is unknown. Hypothesis : Down-regulation of SM22 may actively contribute to arterial pathogenesis. Methods : Five Sm22 knockout ( Sm22 −/− ) mice and their wild type littermates were subjected to carotid denudation, an artery injury model. Analyses were conducted on carotids two weeks after injury. SM22 knockdown was performed in triplicate using siRNA in a VSMC line, PAC1, followed by downstream experiments 3 days after transfection. Results : Sm22 −/− mice developed both enhanced arterial inflammatory response and prominent medial osteochondrogenesis along with remarkable NF- κ B activation compared to their wild-type littermates. The inflammation was evidenced by excessive artery swelling, macrophage infiltration and high induction of pro-inflammatory molecules including COX-2, MCP-1, VCAM-1, ICAM-1 and CX3CL1. The medial osteochondrogenesis was characterized by augmented expression of type II collagen, aggrecan, osteopontin, BMP2, SOX9 and RUNX2. In accordance with the in vivo findings, expression of the aforementioned pro-inflammatory genes and two key osteochondrogenic transcription factors Sox9 and Runx2 was up-regulated after SM22 knockdown in PAC1 cells. Interestingly, SM22 knockdown also led to NF- κ B activation, and inhibition of NF- κ B pathway reversed the up-regulation of both pro-inflammatory genes and osteochondrogenic genes. As an initiator of NF- κ B activation, reactive oxygen species (ROS) production was boosted after SM22 knockdown, and ROS scavenging effectively blocked NF- κ B activation and induction of these genes. Further, the compromised actin cytoskeleton after SM22 knockdown might contribute to the elevated ROS. Conclusions : These findings suggest that loss of SM22 in VSMCs coupled injury induced arterial inflammation with osteochondrogenesis in part via ROS induced NF- κ B activation and that SM22 plays both anti-inflammatory and anti-osteochondrogenic roles in arterial diseases partly by maintaining actin cytoskeleton integrity.

Urotensin II Induced Signal Involved in Proliferation of Vascular Smooth Muscle Cells

Urotensin II receptor (UIIR), bound by the ligand Urotensin II (UII), generates second messengers IP3 and DAG, which stimulates the subsequent release of calcium (Ca2+) in vascular smooth muscle cells (VSMC). Ca2+ influx leads to the activation of Ca2+ dependent kinases via calmodulin binding, resulting in cellular proliferation. We hypothesize that UII signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/ CaM dependent kinases leading to cellular proliferation. UII exposure to Pac1 cell cultures increased intracellular Ca2+, which subsequently activated Ca2+/CaM dependent kinases (CaMKK & CaMKI), ERK 1/ 2, and PKD (PKC‐µ). Pac1 and PAVSMC cells treated with UII resulted in increased proliferation as measured by 3H thymidine uptake. UII‐induced increase of 3H thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that UII stimulation of smooth muscle cells leads to cellular proliferation, utilizing signaling mechanisms involving Ca2+/CaM dependent kinases.

Transforming Growth Factor-β1-induced Expression of Smooth Muscle Marker Genes Involves Activation of PKN and p38 MAPK

Differentiated vascular smooth muscle cells (SMCs) exhibit a work phenotype characterized by expression of several well documented contractile apparatus-associated proteins. However, SMCs retain the ability to de-differentiate into a proliferative phenotype, which is involved in the progression of vascular diseases such as atherosclerosis and restenosis. Understanding the mechanisms involved in maintaining SMC differentiation is critical for preventing proliferation associated with vascular disease. In this study, the molecular mechanisms through which transforming growth factor-beta1 (TGF-beta1) induces differentiation of SMCs were examined. TGF-beta1 stimulated actin re-organization, inhibited cell proliferation, and up-regulated SMC marker gene expression in PAC-1 SMCs. These effects were blocked by pretreatment of cells with either HA1077 or Y-27632, which inhibit the kinases downstream of RhoA. Moreover, TGF-beta1 activated RhoA and its downstream target PKN. Overexpression of active PKN alone was sufficient to increase the transcriptional activity of the promoters that control expression of smooth muscle (SM) alpha-actin, SM-myosin heavy chain, and SM22alpha. In addition, PKN increased the activities of serum-response factor (SRF), GATA, and MEF2-dependent enhancer-reporters. RNA interference-mediated inhibition of PKN abolished TGF-beta1-induced activation of SMC marker gene promoters. Finally, examination of MAPK signaling demonstrated that TGF-beta1 increased the activity of p38 MAPK, which was required for activation of the SMC marker gene promoters. Co-expression of dominant negative p38 MAPK was sufficient to block PKN-mediated activation of the SMC marker gene promoters as well as the serum-response factor, GATA, and MEF2 enhancers. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-beta1 activates PKN to promote differentiation of SMCs.

Smooth muscle--specific expression of CYP4A1 induces endothelial sprouting in renal arterial microvessels.

Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid omega-hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell-specific promoter SM22alpha (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). Beta-galactosidase expression was detected in Ad-SM22-nLacZ-transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1-transduced A7r5 and PAC1 cells. Ad-SM22-4A1-transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79+/-1.77 versus 0.97+/-0.57 nmol 12-hydroxy lauric acid/10(6) cells per h). Smooth muscle-specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04+/-0.29 and 13.43+/-2.84 ng/mg protein in Ad-SM22-nLacZ-transduced and Ad-SM22-4A1-transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63+/-3.66 mm in Ad-SM22-4A1-transduced vessels versus 1.79+/-0.89 mm in Ad-SM22-nLacZ-transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle-specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.

Tumor suppressor MDA-7/IL-24 selectively inhibits vascular smooth muscle cell growth and migration

Abnormalities in smooth muscle cell (SMC) proliferation and differentiation underlie the pathogenesis of proliferative vascular diseases. MDA-7 (HUGO approved symbol IL24) is a unique gene, originally identified as a tumor suppressor and more recently shown to have cytokine activity. MDA-7/IL24 has been implicated in apoptosis and cellular differentiation in tumor cells and in tumor invasion/metastasis in clinical specimens—properties central to SMC remodeling during proliferative vascular diseases. In this study, we evaluated the effects of overexpressing MDA-7/IL24 in various SMC: the apparently “normal” rat PAC1 cell line, primary human coronary artery SMC, and normal rat aortic SMC. We transduced SMC with adenovirus-mda7 (Ad-mda7) or control virus (Ad-Luc) and assessed cell viability, apoptosis, and migration. Ad-mda7 suppressed PAC1 cell growth in a dose-dependent manner while having no effect on normal primary human coronary artery cells or rat aortic SMC, despite strong expression of the MDA-7 transgene in all SMC. Similarly, Ad-mda7 treatment induced apoptosis in PAC1 cells with essentially no effect on normal coronary and rat aortic SMC. Ad-mda7 also inhibited serum-stimulated PAC1 cell migration. Karyotype analysis of PAC1 cells revealed that they exhibit multiple chromosomal aberrations. Importantly, recombinant MDA-7 did not elicit cell death or STAT-3 activation in PAC1 SMC, suggesting that the effects of Ad-mda7 were mediated through an intracellular pathway. These data demonstrate that Ad-mda7 exhibits selectivity in apoptosis induction and growth suppression in an atypical SMC line, raising new questions pertaining to heterogeneity in SMC death susceptibility.

A 27 bp cis-acting sequence is essential for L-type calcium channel α 1C subunit expression in vascular smooth muscle cells

Expression of L-type calcium channels in cardiac myocytes and vascular smooth muscle cells (VSMC) critically regulates the contractile state of these cells. In order to discover the elements in the promoter region of the Ca v1.2 gene encoding the vascular/cardiac calcium channel α 1C subunit that are important for the basal gene expression, ∼2 kb of the 5′-flanking sequence of the Ca v1.2 gene has been cloned in our lab. In this study, using various lengths of the 5′-flanking DNA fused with a luciferase gene as a reporter, we have defined a 493-bp fragment of the cis-regulatory DNA which carries the majority of promoter activity in pulmonary artery smooth muscle (PAC1) cells. DNase I footprinting analysis of this 493-bp DNA using nuclear extracts from PAC1 cells revealed a 27-bp DNA sequence that contains a c-Ets like motif (CAGGATGC). Mutation of the Ets-like site and the respective flanking sequence within the DNase I footprinting protection region induced a marked change in the promoter activity in PAC1 cells. Electrophoretic mobility shift assays (EMSA) confirmed the presence of specific binding factor(s) in PAC1 cells' nuclear extracts for this 27-bp DNA. Competition studies with the wild-type and mutated DNA fragments established the importance of the 27 bp DNA sequence for high-affinity binding of the nuclear proteins to the promoter. We conclude that there is a 27 bp region in the promoter of the Ca v1.2 gene to which nuclear proteins from VSMC bind and strongly regulate the basal promoter activity.

Vascular smooth muscle cells spontaneously adopt a skeletal muscle phenotype: a unique Myf5(-)/MyoD(+) myogenic program.

Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity.(J Histochem Cytochem 48:1173-1193, 2000)

Heterogeneity in the response of vascular smooth muscle to heparin: altered signaling in heparin-resistant cells.

Vascular smooth muscle cells show phenotypic heterogeneity in vivo that affects the extent to which they respond to the antimitogenic effects of heparin. In vitro, heparin-resistant cells are readily selected. This study was undertaken to determine whether differences in the antiproliferative response to heparin involve differences in activity of heparin-sensitive signal transduction pathways. Rat thoracic aorta smooth muscle cells (ASMC) at early passage together with two established vascular smooth muscle lines, PAC-1 and A10, were examined before and after selection for growth in the presence of heparin (10 micrograms/ml). Cells were rendered quiescent and then stimulated with serum. The three cell types showed different sensitivities to the antimitogenic effects of heparin. With respect to [3H]thymidine incorporation, A10 cells were insensitive to 1 microgram/ml heparin whereas PAC-1 cells responded down to 0.05 microgram/ml and ASMC were of intermediate sensitivity. ASMC and PAC-1 cells but not A10 showed a decrease in c-fos mRNA in response to 1 microgram/ml heparin, and a decrease in the c-Fos content of AP-1 DNA binding activity. None of the cells had decreased c-jun mRNA in the presence of heparin. Although induction of c-fos by serum is thought to signal through the Erk mitogen activated protein kinase family, Erk activity was decreased more by 1 microgram/ml heparin in A10 cells than in PAC-1 or ASMC. When cells were selected by growth in the presence of 10 micrograms/ml heparin, A10 cells were unaffected but PAC-1 and ASMC showed a blunted effect of heparin on serum stimulation. In contrast to A10 and their controls not exposed to continuous heparin, heparin-selected PAC-1 and ASMC showed a diminished ability to induce c-fos in response to serum. Smooth muscle cell lines show different responses to the antimitogenic effects of heparin that correlate with the heparin sensitivity of c-Fos/c-Jun expression. Although Erk is implicated in c-fos induction, cells comparatively resistant to heparin still show heparin-dependent inhibition of Erk activation, suggesting that other pathways may be more important for heparin resistance. Furthermore, cells selected for heparin resistance may develop c-fos-independent pathways for proliferation.

Characterisation of the promoter which regulates expression of a phosphoglucomutase-related protein, a component of the dystrophin/utrophin cytoskeleton predominantly expressed in smooth muscle.

We have recently characterised a 60-kDa muscle-specific phosphoglucomutase-related protein (PGM-RP) which is expressed predominantly in adult visceral and vascular smooth muscle. Here we show that the adult vascular smooth muscle cell line PAC1, which retains the capacity to synthesise metavinculin (a marker of the contractile phenotype) also expressed PGM-RP. However, an embryonic smooth muscle cell line A10, which lacks metavinculin, expressed low levels of PGM-RP. Levels of PGM-RP increased in quiescent PAC1 and A10 cells, and were elevated in response to angiotensin II. PGM-RP is therefore a good marker of the contractile/differentiated smooth muscle phenotype. We have sequenced 1.8 kb of the human PGM-RP promoter and shown that it lacks a conventional TATA box. There are multiple transcription start sites, the most predominant of which are inside an initiator sequence (Inr), which is close to two CT boxes and a GATA element. A minimal promoter-CAT construct (p57-CAT) containing the Inr, a CT box and GATA element directed high-level chloramphenicol acetyltransferase (CAT) expression in the differentiated smooth muscle cell line PAC1, and low-level expression in the embryonic smooth muscle cell line A10. This fits well with the pattern of expression of the endogenous gene. A construct (p146-CAT) containing all of the mRNA initiation sites directed a reduced level of CAT expression, and constructs containing 1.8 kb and 3.3 kb upstream of the major transcription start site displayed even lower activity. Sequence comparisons suggest that the PGM-RP promoter evolved from the main phosphoglucomutase promoter which is active in wide range of cell types. The PGM-RP promoter may have acquired negative regulatory elements as expression of the gene became muscle-specific.

Immediate-early gene expression in response to hypertrophic and proliferative stimuli in pulmonary arterial smooth muscle cells.

Remodeling of the pulmonary vascular tree in pulmonary hypertension is associated with hypertrophy and proliferation of smooth muscle cells. Since the stimuli and signaling pathways for these processes are not well understood, we used a rat pulmonary arterial smooth muscle cell line (PAC1) to examine the effects of thrombin and platelet-derived growth factor (PDGF) on cellular growth and immediate-early gene expression. Over 72 h, thrombin (1 unit/ml) caused hypertrophy as reflected by a 102 +/- 12% increase in protein synthesis and a 49 +/- 11% increase in protein content per cell, but no change in cell number. PDGF (2.5 ng/ml) stimulated proliferation as evidenced by an increase in cell number (doubling in 5 days), but no significant change in protein content per cell. Immediate-early gene expression was examined by Northern blotting: both thrombin and PDGF induced egr-1, c-fos, c-jun, junB, and fra-1 mRNAs within 15 min; the response was maximal at 30-60 min (increases ranging from 2.9- to 9.3-fold over control serum-deprived cells) and returned to base-line levels within 2-4 h. Neither agent affected junD mRNA levels. However, thrombin but not PDGF, caused an increase in fosB mRNA levels (7.7 +/- 4.0-fold higher than control, n = 12, p < 0.0005). The immediate-early gene response to both agonists was generally dependent on extracellular Ca2+, Na2+/H+ exchange, and protein kinase C activation, but not on cAMP. The exception was c-jun mRNA, the levels of which were not affected by inhibition of protein kinase C, but decreased significantly by prevention of cAMP formation. Thapsigargin-sensitive intracellular Ca2+ stores were necessary for the response to thrombin, but not to PDGF. These results demonstrate that thrombin is a hypertrophic agent and that PDGF is a proliferative agent in PAC1 cells. These two agonists stimulate increases in a variety of immediate-early gene mRNAs, but only thrombin induces fosB mRNA.

Agonist-selective regulation of polyphosphoinositide metabolism in pulmonary artery smooth muscle cells.

Using a rat pulmonary artery smooth muscle cell line (PAC1), detailed analysis of polyphosphoinositide (PPI) metabolism reveals receptor type-selective patterns in the formation of inositol phosphates and 3-hydroxyphosphorylated PPIs. Responses to several agonists that stimulate hypertrophy or proliferation were examined, and distinct categories of response profile were observed. Thrombin and angiotensin II stimulated the hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and the formation of several cytosolic species of inositol phosphates without the activation of PI 3-hydroxykinase. The response to thrombin was distinctive because a very large production of inositol 1,4-bisphosphate was accompanied by hydrolysis of PI 4-phosphate. The response to platelet-derived growth factor (PDGF) was distinguished by the production of the PI 3-hydroxykinase product, PI 3,4,5-trisphosphate, and the appearance of PI 3-hydroxykinase activity in immunoprecipitates. PDGF treatment of PAC1 cultures did not produce accumulation of detectable amounts of inositol 1,4,5-trisphosphate, although a small sustained elevation in the level of inositol monophosphate and a gradual accumulation of inositol 1,3,4-trisphosphate were observed. Characterization of these distinctive responses permitted us to correlate agonist-regulated PPI metabolism with induction of immediate-early genes and stimulation of hypertrophy or proliferation of PAC1 cultures (Rothman, A., Wolner, B., Button, D., and Taylor, P. (1994) J. Biol. Chem. 269, 6399-6404). Thrombin-stimulated PPI turnover and the production of a high level of inositol bisphosphate may be early signals linked to the induction of fosB and PAC1 cell hypertrophy, whereas the activation of PI 3-hydroxykinase and the accumulation of PI 3,4,5-trisphosphate in response to PDGF appear to be associated with mitogenesis.

Effect of stretch on growth and collagen synthesis in cultured rat and lamb pulmonary arterial smooth muscle cells.

There are no studies of the effect of stretch in cultured pulmonary vascular smooth muscle, and some data suggest that a stretch-mediated increase in connective tissue synthesis in pulmonary arteries is mediated by the endothelium. To investigate whether stretch can serve as a growth stimulus in this smooth muscle, we studied two types of cultured pulmonary arterial smooth muscle cells (a multiply passaged clonal line of rat cells [PAC1], and early passage lamb cells [EPTC]). Cells were grown on a collagen-coated silicone surface and subjected to repetitive stretch (0.33-0.5 Hz; 10-20% strain). The relative rates of total RNA, DNA, protein, and soluble collagen synthesis were determined using 3H precursors, and c-fos and collagen mRNAs by Northern blot analysis. Stretch caused no significant change in the rate of RNA synthesis in either PAC1 cells (+9%) or EPTC (-3%). The relative rate of total protein synthesis was decreased by stretch (6% in PAC1 cells and 36% in EPTC [both NS]) as was the rate of collagen synthesis (-24% in EPTC [NS]). In EPTC, the percentage of 3H-thymidine labeled cells was modestly increased with 24 h stretch (17 +/- 5.7%; P < .001), but trichloroacetic acid (TCA) precipitated 3H-thymidine was unaltered by stretch, and the number of cells not significantly changed with stretch. c-fos mRNA expression was only inconsistently induced by stretch x 30 min in EPTC, and not at all in PAC1 cells. Expression of mRNA for alpha 1 (I) and alpha 1 (III) collagen was not changed significantly by 24 h or 48 h of stretch. We conclude that stretch does not serve as a significant growth stimulus in cultured pulmonary vascular smooth muscle cells in this system. These findings do not rule out the possibility that stretch is a growth stimulus for these cells under different conditions, but do suggest that other models will be needed to determine if and how mechanical stimuli affect growth of pulmonary vascular smooth muscle.