Expression Of Caldesmon Research Articles

Caldesmon expression is decreased in women with anterior vaginal wall prolapse: a pilot study

The purpose of this study is to compare vaginal caldesmon expression in women with and without anterior vaginal wall prolapse. Vaginal tissues were sampled in women with (n = 11) or without (n = 11) vaginal wall prolapse. Caldesmon messenger RNA (mRNA) expression was assessed by quantitative real-time polymerase chain reaction. Immunohistochemistry and digital image analysis were used to determine caldesmon protein expression in the histologic sections. There were no significant differences in demographic data between the two groups. Caldesmon mRNA expression was significantly decreased in the vaginal tissue from women with anterior vaginal wall prolapse compared to women without prolapse [(caldesmon mean +/- SD mRNA expression in relative units) 0.03 +/- 0.03 vs 0.17 +/- 0.17, P = 0.02]. The fractional area of nonvascular caldesmon staining in the vagina of women with anterior vaginal wall prolapse was significantly decreased compared to women without prolapse [mean +/- SD (0.09 +/- 0.04 vs 0.16 +/- 0.09, P = 0.03)]. Vaginal caldesmon expression is significantly decreased in women with anterior vaginal wall prolapse compared to normal subjects.

Coronary artery vascular smooth muscle‐specific contractile protein expression in Syndrome X

A key aspect of coronary collateral growth (CCG) is outward vascular remodeling. We have shown that CCG was impaired in a model of Syndrome X (JCR rat). Outward remodeling is associated with differentiated vascular smooth muscle (VSM), characterized by high VSM‐specific contractile protein expression, vs. inward remodeling, associated with de‐differentiated VSM, characterized by loss of VSM‐specific proteins. We hypothesized that expression of VSM‐specific contractile proteins was significantly lower in Syndrome X. SM‐alpha‐actin, SM‐myosin, smoothelin and caldesmon expression was evaluated by immunohistochemistry and Western blot in coronary arteries (CA) of JCR vs. healthy (WKY) rats. Results show decreased SM‐myosin (25%), SM‐alpha‐actin (50%), smoothelin (30%) and caldesmon (48%) expression in JCR vs. WKY CA. The pattern of expression differed between large (lCA) vs. small (sCA) arteries. Caldesmon was consistently lower in lCA and sCA of JCR rats, but SM‐alpha‐actin and smoothelin exhibited significantly larger decreases in sCA vs. lCA, while SM‐myosin was significantly decreased in lCA but not in sCA. These results demonstrate that coronary artery VSM is de‐differentiated in Syndrome X, but the extent of specific contractile protein downregulation differs between large vs. small coronary arteries, and may provide a partial explanation for impaired CCG in Syndrome X. NIH HL093052

Evaluating the differentiation state of aortic vascular smooth muscle cells in the metabolic syndrome

The JCR rat, a model mimicking human metabolic syndrome exhibits altered vascular remodeling. These features make the JCR rat an excellent model for study of vascular smooth muscle cell (VSMC) phenotype switching. To date, basal contractile protein expression in VSMCs from JCR rats has not been determined. We hypothesized that the expression profile in JCR VSMCs would be indicative of a de‐differentiated, synthetic phenotype compared to VSMCs from healthy WKY rats, resulting in decreased expression of SM‐specific contractile proteins, and increased VSMC proliferation and migration. We isolated aortic VSMCs from JCR and WKY rats and compared basal contractile protein expression by Western blotting. Results indicate that SM‐myosin heavy chain and SM‐alpha‐actin expression was decreased 46% and 76%, respectively, in JCR vs. WKY VSMCs, caldesmon expression was six fold higher, and smoothelin levels remained unchanged. Proliferation rate, determined by BrdU incorporation, was increased 63% in JCR VSMCs. In response to scratch wounding, JCR VSMCs exhibited significantly greater migration than WKY VSMCs. These findings demonstrate that cultured JCR aortic VSMCs display a more de‐differentiated phenotype when compared to WKY aortic VSMC, which may underlie altered vascular remodeling in the metabolic syndrome. NIH HL 084159 and HL 093052

Caldesmon is essential for cardiac morphogenesis and function: In vivo study using a zebrafish model

The zebrafish homologue of caldesmon is similar to the mammalian low molecular weight caldesmon ( l-CaD). In this study, we explored the effects of caldesmon knockdown on vertebrate heart development in vivo. In a zebrafish model caldesmon was knocked down resulting in defective cardiac morphogenesis, muscularization and function. The data provide the first functional assessment of the role of caldesmon in cardiac development in vivo, and indicate that caldesmon is essential for proper cardiac organogenesis and function. Because caldesmon expression remarkably influences cardiac muscularization, the findings are relevant for designing future therapeutic strategies in the regeneration of cardiac damage.

Glucocorticoid Receptor-mediated Expression of Caldesmon Regulates Cell Migration via the Reorganization of the Actin Cytoskeleton

Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.

Effect of long-term partial bladder outlet obstruction on caldesmon isoforms and their correlation with contractile function

In the present study, we investigate the expression of caldesmon (CAD) isoforms in rabbit detrusor smooth muscles (DSM) during the progression of partial bladder outlet obstruction and relate them with the time course of obstruction. Detrusor samples were obtained from the bladders of rabbits with partial bladder outlet obstruction and sham-operated control rabbits after 1, 2, 4, and 8 weeks of obstruction. Contractile responses to field stimulation and carbachol were determined in the isolated bladder strips. Western blotting was used to determine the relative levels of CaD isoform expression at the protein levels. The contractile responses decreased progressively over the course of obstruction. The expression of l-CaD increased significantly to approximately the same extent as the 1-4-week obstructed groups and further in the 8-week obstructed group. The expression of h-CaD increased in all of the obstructed bladders, but at significantly higher levels in the 1-2-week obstructed bladders compared to the control and 4-8-week obstructed bladders. The changes in the isoforms of CaD may be part of the molecular mechanism for bladder compensation following partial bladder outlet obstruction. The overexpression of l-CaD and the h-CaD/l-CaD ratio could be markers for the status of DSM remodeling and dysfunction.

Caldesmon suppresses cancer cell invasion by regulating podosome/invadopodium formation

The podosome and invadopodium are dynamic cell-adhesion structures that degrade the extracellular matrix (ECM) and promote cell invasion. We recently reported that the actin-binding protein caldesmon is a pivotal regulator of podosome formation. Here, we analyzed the caldesmon’s involvement in podosome/invadopodium-mediated invasion by transformed and cancer cells. The ectopic expression of caldesmon reduced the number of podosomes/invadopodia and decreased the ECM degradation activity, resulting in the suppression of cell invasion. Conversely, the depletion of caldesmon facilitated the formation of podosomes/invadopodia and cell invasion. Taken together, our results indicate that caldesmon acts as a potent repressor of cancer cell invasion.

Two-dimensional electrophoresis analyses of atopic dermatitis and the chances to detect new candidate proteins by the variations in immobilized pH gradient strips

Proteomic approaches, one of the high-throughput technologies, have been used to search for the proteins that are abnormally expressed in human diseases. The atopic dermatitis (AD)-associated genes or proteins are gradually being reported. In accordance with recent reports, we conducted the serial proteomic studies to compare with the protein expression level and to find a critical protein associated with AD. We applied two-dimensional electrophoresis (2-DE) coupled with MADI-TOF as well as LC-MS/MS to detect dysregulated protein in the AD proteome obtained from the patient-derived primary cells. Real-time PCR was also conducted to compare with the proteomic results in the transcriptional level. We successfully detected new AD-associated proteins in the AD-derived fibroblasts 2D-PAGE studies due to the IPG strip variations after conducting MALDI-TOF mass spectrometry and identification of the proteins. From the real-time PCR quantifications, we found that the altered expression of caldesmon 1 isoform 5, nucleophosmin 1, esterase D and chloride intracellular channel 4 were well matched both at the transcriptional and translational levels, and this suggested that these proteins may have important involvement with the pathogenesis of AD. By simply repeating the trials with changing the commercial strips with different lot numbers in the 2D-PAGE analysis, this provided us with new finding in the AD-derived samples. This approach we used may increase the chances of finding new candidate proteins in the clinical samples and it may also be applicable for proteomic studies of other diseases.

Development of Immunoglobulin A Nephropathy- Like Disease in β-1,4-Galactosyltransferase-I-Deficient Mice

Beta4 galactosylation of glycoproteins plays important roles in protein conformation, stability, transport, and clearance from the circulation. Recent studies have revealed that aberrant glycosylation causes various human diseases. Here we report that mice lacking beta-1,4-galactosyltransferase (beta4GalT)-I, which transfers galactose to the terminal N-acetylglucosamine of N- and O-linked glycans in a beta-1,4 linkage, spontaneously developed human immunoglobulin A nephropathy (IgAN)-like glomerular lesions with IgA deposition and expanded mesangial matrix. beta4GalT-I-deficient mice also showed high serum IgA levels with increased polymeric forms as in human IgAN. IgAN is the most common form of glomerulonephritis, and a significant proportion of patients progress to renal failure. However, pathological molecular mechanisms of IgAN are poorly understood. In humans, abnormal character of serum IgA, especially serum IgA1 with aberrant galactosylation and sialylation of O-glycans in its hinge region is thought to contribute to the pathogenesis of IgAN. Mouse IgA has N-glycans but not O-glycans, and beta4-galactosylation and sialylation of the N-glycans on the serum IgA from beta4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We propose that carbohydrates of serum IgA are involved in the development of IgAN, whether the carbohydrates are O-glycans or N-glycans.

Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells

Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell–cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell–matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24–48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40–50% of the population of caldesmon-expressing cells demonstrated high levels of GFP–caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca ++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 μm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell–cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell–matrix adhesions, and destabilization of cell–cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.

Caldesmon is an integral component of podosomes in smooth muscle cells

Podosomes are highly dynamic actin-based structures commonly found in motile and invasive cells such as macrophages, osteoclasts and vascular smooth muscle cells. Here, we have investigated the role of caldesmon, an actin-binding protein, in the formation of podosomes in aortic smooth muscle A7r5 cells induced by the phorbol ester PDBu. We found that endogenous low molecular weight caldesmon (l-caldesmon), which was normally localised to actin-stress fibres and membrane ruffles, was recruited to the actin cores of PDBu-induced podosomes. Overexpression of l-caldesmon in A7r5 cells caused dissociation of actin-stress fibres and disruption of focal adhesion complexes, and significantly reduced the ability of PDBu to induce podosome formation. By contrast, siRNA interference of caldesmon expression enhanced PDBu-induced formation of podosomes. The N-terminal fragment of l-caldesmon, CaD40, which contains the myosin-binding site, did not label stress fibres and was not translocated to PDBu-induced podosomes. Cad39, the C-terminal fragment housing the binding sites for actin, tropomyosin and calmodulin, was localised to stress fibres and was translocated to podosomes induced by PDBu. The caldesmon mutant, CadCamAB, which does not interact with Ca2+/calmodulin, was not recruited to PDBu-induced podosomes. These results show that (1) l-caldesmon is an integral part of the actin-rich core of the podosome; (2) overexpression of l-caldesmon suppresses podosome formation, whereas siRNA knock-down of l-caldesmon facilitates its formation; and (3) the actin-binding and calmodulin-binding sites on l-caldesmon are essential for the translocation of l-caldesmon to the podosomes. In summary, this data suggests that caldesmon may play a role in the regulation of the dynamics of podosome assembly and that Ca2+/calmodulin may be part of a regulatory mechanism in podosome formation.

TGFβ Induces Loss of Epithelial Character and Smooth Muscle Cell Differentiation in Epicardial Cells

During embryogenesis, epicardial cells undergo epithelial-mesenchymal transformation (EMT), invade the myocardium, and differentiate into components of the coronary vasculature including smooth muscle cells. We tested the hypothesis that transforming growth factor β (TGFβ) stimulates EMT and smooth muscle differentiation of epicardial cells. To test this hypothesis, we cultured explanted epicardium from 4 days in ovo chick hearts on three dimensional collagen gels or collagen coated chamber slides. In epicardial explants, TGFβ1 and TGFβ2 induce loss of epithelial morphology, cytokeratin and membrane-associated Zonula Occludens-1 (ZO-1), and increase the smooth muscle markers, calponin and caldesmon. TGFβ activates a family of membrane bound serine-threonine kinases, including activin receptor-like kinase (ALK) 5, resulting in the stimulation of a number of downstream effectors. Inhibition of ALK5 kinase activity blocks these effects while overexpression of constitutively active (ca) ALK5 increases cell invasion by 42%. Overexpression of Smad 3 did not mimic the effects of caALK5. Inhibition of p160 rho kinase or p38 MAP kinase prevented the loss of epithelial morphology in response to TGFβ while only inhibition of p160 rho kinase blocked TGFβ–stimulated caldesmon expression. These data demonstrate that TGFβ stimulates loss of epithelial character and smooth muscle differentiation in epicaridal cells via a mechanism that requires ALK5 and p160 rho kinase. NIH HL67105; Vanderbilt MSTP 5 T32 GM07347.

Transforming growth factor‐β induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells

During embryogenesis, epicardial cells undergo epithelial-mesenchymal transformation (EMT), invade the myocardium, and differentiate into components of the coronary vasculature, including smooth muscle cells. We tested the hypothesis that transforming growth factor-beta (TGFbeta) stimulates EMT and smooth muscle differentiation of epicardial cells. In epicardial explants, TGFbeta1 and TGFbeta2 induce loss of epithelial morphology, cytokeratin, and membrane-associated Zonula Occludens-1 and increase the smooth muscle markers calponin and caldesmon. Inhibition of activin receptor-like kinase (ALK) 5 blocks these effects, whereas constitutively active (ca) ALK5 increases cell invasion by 42%. Overexpression of Smad 3 did not mimic the effects of caALK5. Inhibition of p160 rho kinase or p38 MAP kinase prevented the loss of epithelial morphology in response to TGFbeta, whereas only inhibition of p160 rho kinase blocked TGFbeta-stimulated caldesmon expression. These data demonstrate that TGFbeta stimulates loss of epithelial character and smooth muscle differentiation in epicardial cells by means of a mechanism that requires ALK5 and p160 rho kinase.

Nuclear Factor of Activated T Cells and Serum Response Factor Cooperatively Regulate the Activity of an α-Actin Intronic Enhancer

Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.

OVER EXPRESSION OF SMOOTH MUSCLE THIN FILAMENT ASSOCIATED PROTEINS IN THE BLADDER WALL OF DIABETICS

The thin filament associated proteins caldesmon, tropomyosin and calponin have been shown to modulate actin-myosin interaction, actomyosin adenosine triphosphatase and contraction in smooth muscle. This study was performed to determine whether the expression of these proteins is altered in diabetes induced decrease in the contractility of bladder wall smooth muscle. Detrusor samples were obtained from New Zealand White male rabbits with alloxan induced diabetes, and from age and sex matched control rabbits. In addition, a bladder myocyte cell line, which continues to express smooth muscle phenotype, was exposed to either normal (5 mM) or high (50 mM) concentrations of glucose. The levels of expression of the thin filament associated proteins were determined at the mRNA and protein levels by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. Detrusor smooth muscle tissue from rabbits with alloxan induced diabetes showed over expression of thin filament associated proteins, calponin, tropomyosin and caldesmon when compared with that of the control. Similar up-regulation was seen also in bladder myocytes in cultures treated with 50 mM glucose, indicating that the high glucose induced the changes. Our results suggest that the increased expression of thin filament proteins, calponin, tropomyosin and caldesmon in diabetic rabbits might alter the contractile and cytoskeletal structure in bladder myocytes. The over expression of these thin filament associated proteins, which suppresses actin-myosin interaction and actomyosin adenosine triphosphatase, and the enhancement of this suppression by tropomyosin are likely to have an effect on the relationship between force and myosin light chain phosphorylation, requiring higher levels of phosphorylation in diabetic detrusor compared with that of control. The downstream effects of high glucose (eg oxidative stress) appear to modulate the transcriptional regulation of thin filament mediated regulatory proteins in bladder smooth muscle.

Specific disruption of smooth muscle caldesmon expression in mice

Caldesmon (CaD) is an actin-binding protein that is capable of inhibiting the actomyosin ATPase activity in vitro. CaD has a single gene that is alternatively spliced to generate the smooth muscle-specific form, h-CaD, and a shorter isoform, l-CaD, that is present only in non-muscle cells. The difference between h- and l-CaD is a highly charged repeating sequence, corresponding to a 35 nm-long single helical region that separates the N-terminal domain from the C-terminal domain of h-CaD. To test whether such an elongated h-CaD is essential for smooth muscles to function properly, we have specifically abrogated its expression in the mouse by targeting h-CaD without affecting the expression of l-CaD. After genotyping, we have obtained homozygous knockout mice that indeed lack h-CaD, but nevertheless express varying amounts of l-CaD in a tissue-dependent fashion. The contractility of smooth muscles isolated from the knockout animals is currently under investigation.

Characterization of smooth muscle cell and pericyte differentiation in the rat retina in vivo.

To identify and apply a range of suitable mural cell markers and undertake an in vivo characterization of pericyte and smooth muscle cell (SMC) differentiation in the developing rat retina. Pericyte and SMC differentiation was characterized by immunohistochemistry with antibodies to NG2, desmin, alpha-smooth muscle actin (SMA), calponin, and caldesmon. Immature mural precursor cells (MPCs) were scattered throughout the primitive capillary plexus in the rat retina at embryonic day (E)20. The postnatal differentiation of pericytes and arteriolar and venous SMCs followed with distinct intermediate phenotypes. SMC differentiation coincided with selection of major vessels from the primordial capillary bed. Maturation of radial arteriolar SMCs was indicated by the expression of calponin and caldesmon, proteins that play a role in the regulation of SMC contraction. The mere presence of immature mural cells did not confer vessel stability; rather vessel stability in the developing rat retina coincided with caldesmon and calponin expression in arteriolar SMCs. This normative data and the identification of suitable in vivo markers of pericytes and SMCs will allow meaningful interpretation of the changes in these cell types. When examining the role of mural cells in developmental and pathologic vascularization, the results show that there is a need to use multiple-marker immunohistochemistry because of significant mural cell heterogeneity. The observation that the expression of caldesmon and calponin in arteriolar SMCs coincides with resistance to hyperoxia in the developing rat retina, lead us to suggest that maturation of SMCs and their consequent ability to regulate blood flow may play a key role in vessel stabilization.

Differential Expression of Splicing Variants of the Human Caldesmon Gene ( CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

Caldesmon is a cytoskeleton-associated protein which has not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were pre-screened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 + 4, and 1' + 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, mutation scanning was performed by a coupled in vitro transcription/translation assay (IVTT). No premature stop mutations were traced by the IVTT. The transcriptional changes consequently resulted in up-regulation at the protein expression level. The up-regulated expression of caldesmon was coincident with the down-regulated expression of tight junction proteins (occludin and ZO-1). The results support the notion that missplicing of the CALD1 gene in glioma microvasculature is an independent epigenetic event regulated at the transcriptional level. The event coexists with tight junction (TJ) breakdown of the endothelial cells in glioma microvasculature. The data reveal a novel mechanism contributing to dysfunctionality of glioma neovascularization.

Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins.

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC(17b)) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins.

Over Expression of Smooth Muscle Specific Caldesmon by Transfection and Intermittent Agonist Induced Contraction Alters Cellular Morphology and Restores Differentiated Smooth Muscle Phenotype

The thin filament associated protein h-caldesmon (h-CaD) modulates actin myosin interaction and contraction. Bladder outlet obstruction and detrusor hypertrophy are associated with the over expression of the nonmuscle CaD isoform l-CaD. It implies a poorly differentiated state of bladder myocytes and cytoskeletal remodeling in detrusor hypertrophy. We determined if h-CaD expression can be increased in a unique bladder smooth muscle (BSM) cell line derived from obstructed rabbit bladder smooth muscle that over expresses l-CaD. We examined whether the genetic restoration of h-caldesmon is possible in bladder smooth muscle cells by transfection or by agonist mediated contraction and whether this manipulation would alter cellular morphology. BSM cells were transfected with chicken h-CaD cDNA inserted into a mammalian vector. In another experiment BSM cells underwent intermittent bethanechol induced stimulation. h-CaD mRNA and protein were quantified with reverse transcriptase-polymerase chain reaction and Western blot analyses. Cell morphology was assessed using phase, video and confocal microscopy after double immunostaining with antibodies against alpha-actin and caldesmon. Reverse transcriptase-polymerase chain reaction using primers specific for the transfected vector and h-CaD cDNA confirmed stable transfection of cells and increased content of h-CaD mRNA. Following bethanechol induced intermittent contraction Western blotting revealed 80% relative over expression of h-CaD in treated transfected cell lines (p <0.05) and 74% (not significant) in treated nontransfected controls. Confocal immunofluorescence microscopy revealed CaD in the cytoplasmic filaments co-localized to alpha-actin in the main cell body and perinuclear region in transfected cells, in contrast to the diffuse, irregular distribution of these filaments in control cells. A unique bladder myocyte cell line was successfully and stably transfected with h-CaD cDNA. We show that agonist induced intermittent contraction preferentially increases h-CaD expression, the predominant CaD in nonobstructed bladder smooth muscle, and the restoration of h-CaD alters cell morphology and the organization of cytoplasmic filaments in cells derived from obstructed rabbit detrusor musculature.