Interaction of CArG elements and a GC-rich repressor element in transcriptional regulation of the smooth muscle myosin heavy chain gene in vascular smooth muscle cells

We have previously shown that maximal expression of the rat smooth muscle myosin heavy chain (SM-MHC) gene in cultured rat aortic smooth muscle cells (SMCs) required the presence of a highly conserved domain (nucleotides -1321 and -1095) that contained two positive-acting serum response factor (SRF) binding elements (CArG boxes 1 and 2) and a negative-acting GC-rich element that was recognized by Sp1 (Madsen, C. S., Hershey, J. C., Hautmann, M. B., White, S. L., and Owens, G. K. (1997) J. Biol. Chem. 272, 6332-6340). In this study, to better understand the functional role of these three cis elements, we created a series of SM-MHC reporter-gene constructs in which each element was mutated either alone or in combination with each other and tested them for activity in transient transfection assays using primary cultured rat aortic SMCs. Results demonstrated that the most proximal SRF binding element (CArG-box1) was active in the absence of CArG-box2, but only upon removal of the GC-rich repressor. In contrast, regardless of sequence context, CArG-box2 was active only when CArG-box1 was present. We further demonstrated using electrophoretic mobility shift assays that Sp1 binding to the GC-rich repressor element did not prevent SRF binding to the adjacent CArG-box2. Thus, unlike other proteins reported to inhibit SRF activity, the repressor activity associated with the GC-rich element does not appear to function through direct inhibition of SRF binding. As a first step toward understanding the importance of these elements in vivo, we performed in vivo footprinting on the intact rat aorta. We demonstrated that both CArG boxes and the GC-rich element were bound by protein within the animal. Additionally, using the rat carotid injury model we showed that Sp1 protein was significantly increased in SMCs located within the myointimal lesion, suggesting that increased expression of this putative repressor factor may contribute to the decreased SM MHC expression within SMCs found in myointimal lesions.     

Localization of 17-kDa myosin light chain isoforms in cultured aortic smooth muscle cells

Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.     

Effect of controllable stress on myosin heavy chain expression and muscle-specific protection by clomipramine

This study evaluated the influence of a controllable and painless stress, conditioned bright-light active-avoidance, on the expression of myosin heavy chain (MHC) protein isoforms in two nape and three masticatory rat muscles: longissimus capitis (L), rectus capitis dorsalis major (R), anterior digastric (AD), anterior temporalis (AT) and masseter superficialis (MS). The effects of a concomitant antidepressant treatment with clomipramine (CMI) on the muscle structure were also investigated. The three adult fast MHC isoforms were detected in all muscles studied: MHC 2A, 2X and 2B. The AT structure was not significantly modified by stress either under saline or under CMI treatment. In the other muscles studied, the stress situation induced a marked increase in the relative expression of MHC 2B and a decrease in MHCs 2X and 2A, except in L in which the MHC 2A decrease did not reach a statistically significant level. Under controllable stress, the CMI treatment led to the same MHC profile in AT, L, R and AD as saline, except in L where the MHC 2X decrease was no longer statistically significant. However, in MS, under controllable stress and CMI treatment, the MHC distribution was significantly different from the stressed saline-treated group and became comparable to the control again. MHC 2B has a higher shortening velocity than MHC 2X, which has a higher one than MHC 2A. According to total MHC isoform expression, the controllable stress-induced transformations would thus lead to increased velocity of all five muscles studied except in AT. This latter seems, therefore, not very sensitive to environmental requirements. Our results indicate that controllable stress produces important changes in the contractile properties of nape and masticatory muscles. Furthermore, this study demonstrates the protective effect of CMI against muscle structure transformations induced by controllable stress in MS, and that these effects are muscle type-specific.     

The light chain-binding domain of the smooth muscle myosin heavy chain is not the only determinant of regulation

Interactions between the dephosphorylated regulatory light chains (RLCs) of smooth muscle myosin are involved in maintaining the enzymatically "off" state. Expressed chimeric smooth muscle heavy meromyosins containing skeletal muscle myosin heavy chain (HC) sequences were used to assess the relative importance of the light chain-binding domain (or "neck") to regulation. Surprisingly, regulation remained intact with a skeletal RLC-binding site. A chimera with the entire alpha-helical neck composed of skeletal HC sequence showed 2-fold regulation of motility and nearly 5-fold regulation of actin-activated ATPase activity. Complete activation of the dephosphorylated state (i.e. complete loss of regulation) occurred when skeletal HC sequence extended from the head/rod junction to the SH1-SH2 helix. Smooth muscle-specific sequences near the motor domain may therefore position the regulatory domain in a way that optimizes RLC-rod-head interactions, thus enabling a completely off state when the RLC is dephosphorylated. Conversely, a chimera that joins the motor domain from unconventional myosin V to the smooth muscle myosin neck and rod showed only 2-fold regulation. The presence of the smooth muscle light chain-binding region and rod is therefore not sufficient to confer complete phosphorylation-dependent regulation upon all motor domains of the myosin family.     

Interferon gamma up-regulates a novel protein in vascular smooth muscle cells

PURPOSE: By means of the technique of messenger RNA (mRNA) differential display, we previously isolated a partial DNA clone found to be down-regulated at the polytetrafluoroethylene (PTFE) hyperplastic arterial anastomosis compared with the normal artery. The partial DNA gene sequence was found to be homologous with interferon gamma up-regulated protein (IGUP) first found in human psoriatic keratinocytes. We cloned the entire IGUP gene from human vascular smooth muscle cells (VSMCs) to determine its regulation by gamma interferon (gamma-IFN) and other cytokines in cultured human VSMCs. METHODS: By means of polymerase chain reaction, the IGUP gene was amplified from a QUICK-Clone complementary DNA human aorta kit using 5' and 3' oligonucleotide primers to the known IGUP sequence. Immunohistocytochemistry studies compared normal artery and distal anastomotic IH. Human VSMCs were stimulated with 1000 U/mL of gamma-IFN, 5 ng/mL of platelet-derived growth factor BB (PDGF-BB), 3. 2 ng/mL basic fibroblast growth factor, 3.3 ng/mL transforming growth factor beta(TGF-beta), 10 ng/mL of vascular endothelial growth factor, and 10% fetal bovine serum (FBS) for zero, 24, 48 and 72 hours. Western blot analysis of lysates of the stimulated VSMCs was performed to determine up-regulation of IGUP. RESULTS: DNA sequencing confirmed the cloning of the entire coding region of the IGUP gene with 100% homology to the known IGUP DNA sequence. There was strong expression of IGUP in quiescent VSMCs and marked reduction of expression of IGUP in proliferating smooth muscle cells. gamma-IFN was the only cytokine, of the cytokines evaluated, to up-regulate production of IGUP in VSMCs. CONCLUSION: IGUP is a novel protein in VSMCs found to be down-regulated in areas of anastomotic IH, as compared with a normal artery. We have now shown IGUP to be up-regulated only by gamma-IFN in human VSMCs. IGUP may, therefore, be the intermediary for the known gamma-IFN inhibition of human VSMC proliferation.     

Skeletal muscle myosin heavy chain synthesis in type 1 diabetes

Although insulin's anticatabolic effect on protein metabolism in type 1 diabetes has been clearly shown to be related to the inhibition of protein breakdown, insulin's effect on muscle protein synthesis remains controversial. Cross-limb studies and measurements of synthesis rates of mixed muscle protein have yielded conflicting results. These measurements represent the mean synthesis of several muscle proteins and may miss changes in the synthesis rates of individual muscle proteins. We measured the fractional synthesis rates of myosin heavy chain (MHC), the principal muscle contractile protein, and mixed muscle protein (MMP) in six type 1 diabetic patients during insulin deprivation and insulin treatment. Comparisons were made with six healthy control subjects. Muscle biopsies were taken at 2 h and 8 h during a primed continuous infusion of L-[1-13C]leucine. MHC was purified by a preparative continuous elution gel electrophoresis, and fractional synthesis rates were calculated. We found that in type 1 diabetic subjects, the fractional synthesis rates of MHC and MMP during insulin treatment are similar to those of control subjects. Acute insulin deprivation did not affect either the synthesis rate or the ratio of MHC to MMP in type 1 diabetic subjects. In the postabsorptive state, acute insulin deprivation has no effect on MHC or MMP synthesis in type 1 diabetic patients.     

Developmental transitions in the myosin heavy chain phenotype of human respiratory muscle

The renal lymphatic system plays an important role in removing excess fluid from the kidneys. Unfortunately, the factors influencing lymphatic flow are difficult to measure. We used a simple model to represent renal lymphatics as a single pressure source (PL) pushing lymph through a single resistance (RL). In anesthetized dogs, we cannulated renal lymphatics and measured lymph flow rate (QL) as we varied pressure (PO) at the outflow end of the lymphatics. There was no significant change in QL as we increased PO from -5 to 0 cm H2O. In other words, there was a plateau in the QL vs. PO relationship. At higher PO's, QL decreased linearly with increases in PO. From this linear relationship, we calculated RL as -delta PO/ delta QL and we took PL as the PO at which QL = 0 microliter/min. At baseline, RL = 0.34 +/- 0.14 (SD) cm H2O.min/microliter and PL = 8.2 +/- 4.4 cm H2O. When we increased renal venous pressure (PV) from baseline (3.5 +/- 3.0 cm H2O), the plateau in the QL vs. PO relationship extended to higher PO's, RL decreased, and PL increased. Renal interstitial fluid volume and interstitial pressure increased following elevation of PV. The extension of the QL vs. PO plateau with increasing PV suggests that renal interstitial pressure may partially collapse intrarenal collecting lymphatics which may compromise lymph flow.     

Effect of preincubation on smooth muscle myosin light chain kinase activity

Phosphorylation of myosin regulatory light chain (RLC) catalysed by myosin light chain kinase (MLCK) is a key reaction in the regulation of actin-myosin interaction in smooth muscle. The activation of MLCK by calmodulin (CaM) and Ca2+ was investigated over a wide range of the enzyme concentrations using myosin or its RLC with Mw = 20 kDa as substrates. Kinase activation by CaM (at saturating Ca2+ concentrations) was characterized by positive cooperativity even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between MLCK and CaM. The activation of the kinase by Ca2+ was also cooperative but only at relatively low CaM levels. This cooperativity was shown to result from time dependent changes in MLCK that take place during its incubation with Ca2+ and CaM before substrate addition in phosphorylation assays. As a result the kinase activity as a function of its concentration at constant CaM level was biphasic: there was the activity optimum at 1:1 ratio of CaM to MLCK and almost complete inhibition at 3 to 7 molar excess of kinase over CaM. Such changes that take place during 10 to 15 min preincubation with Ca2+ and CaM may involve the kinase supramolecular structure formation or/and its conformational rearrangements.     

Corticosteroids inhibit the expression of the vascular endothelial growth factor gene in human vascular smooth muscle cells

The vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and enhances vascular permeability and edemagenesis. VEGF is also a major regulator of angiogenesis and may be a key target for inhibiting angiogenesis in angiogenesis-associated diseases. Among the extensively studied angiostatic compounds are several corticosteroids when used alone or in combination with heparin. In this study we present evidence for an additional mechanism of action of hydrocortisone, cortisone and dexamethasone in inhibiting edemagenesis or angiogenesis. In cultures of aortic human vascular smooth muscle cells these corticosteroids (1 x 10(-8) to 1 x 10(-12) M) abolished the platelet-derived growth factor-induced (PDGF) expression of the VEGF gene in a dose-dependent manner. In contrast, two precursors of corticosteroids, desoxycorticosterone or pregnenolone, did not affect PDGF-induced VEGF expression. Our findings indicate that the capacity of corticosteroids to reduce edema or to prevent new blood vessel formation may be attributed, at least in part to the ability of these agents to abolish the expression of VEGF.     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

The effects of 14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044) on myosin heavy chain (MHC) isoform content of the rat soleus muscle and single muscle fibers were determined. On the basis of electrophoretic analyses, there was a de novo synthesis of type IIx MHC but no change in either type I or IIa MHC isoform proportions after either SF or HS compared with controls. The percentage of fibers containing only type I MHC decreased by 26 and 23%, and the percentage of fibers with multiple MHCs increased from 6% in controls to 32% in HS and 34% in SF rats. Type IIx MHC was always found in combination with another MHC or combination of MHCs; i.e., no fibers contained type IIx MHC exclusively. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal weight bearing and that the absence of weight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.     

A 340 kDa hyaluronic acid secreted by human vascular smooth muscle cells regulates their proliferation and migration

The formation of atherosclerotic lesions is characterized by invasion of vascular smooth muscle cells (VSMC) into the tunica intima of the arterial wall and subsequently by increased proliferation of VSMC, a process apparently restricted to the intimal layer of blood vessels. Both events are preceded by the pathological overexpression of several growth factors, such as platelet-derived growth factor (PDGF) which is a potent mitogen for VSMC and can induce their chemotaxis. PDGF is generally not expressed in the normal artery but it is upregulated in atherosclerotic lesions. We have previously shown that PDGF-BB specifically stimulates proliferating VSMC to secrete a 340 kDa hyaluronic acid (HA-340). Here, we present evidence regarding the biological functions of this glycan. We observed that HA-340 inhibited the PDGF-induced proliferation of human VSMC in a dose-dependent manner and enhanced the PDGF-dependent invasion of VSMC through a basement membrane barrier. These effects were abolished following treatment of HA-340 with hyaluronidase. The effect of HA-340 on the PDGF-dependent invasion of VSMC coincided with increased secretion of the 72-kDa type IV collagenase by VSMC and was completely blocked by GM6001, a hydroxamic acid inhibitor of matrix metalloproteinases. HA-340 did not exert any chemotactic potency, nor did it affect chemotaxis of VSMC along a PDGF gradient. In human atheromatic aortas, we found that HA-340 is expressed with a negative concentration gradient from the tunica media to the tunica intima and the atheromatic plaque. Our findings suggest that HA-340 may be linked to the pathogenesis of atherosclerosis, by modulating VSMC proliferation and invasion.     

Natriuretic peptide family as a novel antimigration factor of vascular smooth muscle cells

Vascular smooth muscle cell (SMC) migration is proposed to be an important process in the initiation and/or progression of atherosclerosis. The present study examined the effects of the natriuretic peptide family (atrial, brain, and C-type natriuretic peptides; ANP, BNP, and CNP) on the migration of cultured rat SMCs, using Boyden's chamber methods. Fetal calf serum (FCS) and platelet-derived growth factor (PDGF)-BB potently stimulated SMC migration. Rat ANP(1-28), rat BNP-45, and rat CNP-22 clearly inhibited SMC migration stimulated with FCS or PDGF-BB in a concentration-dependent manner. CNP-22 had the most potent inhibitory effect compared with other natriuretic peptides. When PDGF-BB-induced migration was separated into chemotactic and chemokinetic activities, the chemotactic component was strongly inhibited by these natriuretic peptides. Such inhibition by these natriuretic peptides was paralleled by an increase in the cellular level of cyclic GMP. The addition of a cyclic GMP analogue, 8-bromo cyclic GMP, and an activator of the cytosolic guanylate cyclase, sodium nitroprusside, significantly inhibited FCS- and PDGF-BB-stimulated migration in a concentration-dependent manner. These results suggest that natriuretic peptides, especially CNP-22, inhibit FCS- or PDGF-BB-stimulated SMC migration at least in part through a cyclic GMP-dependent process. Thus, the natriuretic peptide family may play a role as an antimigration factor of SMCs under certain circumstances.