Nonmuscle Caldesmon Research Articles

Glucocorticoid receptor-induced non-muscle caldesmon regulates metastasis in castration-resistant prostate cancer

Lethal prostate cancer (PCa) is characterized by the presence of metastases and development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. Here, we report that in vitro downregulation of l-CaD promotes epithelial phenotype and reduces spheroid growth in 3D, which is reflected in vivo in reduced formation of metastases in zebrafish PCa xenografts. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition (EMT) transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets, and GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation.

Abstract A024: Glucocorticoid receptor-induced non-muscle caldesmon regulates growth and metastasis in castration-resistant prostate cancer

Abstract Lethal prostate cancer (PCa) is characterized by the presence of metastases and the development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. The goal of our study was to characterize the potential role of l-CaD in PCa metastases and antiandrogen therapy resistance. In this study, we extracted co-expression data of l-CaD from the largest public PCa patient data sets and identified the common transcripts between the sets. The common co-expression hits were used to recognize biological processes associated with l-CaD in the PCa context. Next, we used in vitro techniques, including 3D culture, cell viability assays, immunofluorescence staining, and Western blotting, to study the role of l-CaD in PCa with a focus on cancer hallmarks and the recognized associated biological processes. Finally, we used in vivo zebrafish and mouse xenograft models to verify the findings observed in silico in PCa patient data and in vitro in PC3, DU145, and VCaP cell lines. Here, we report that in vitro downregulation of l-CaD promotes the epithelial phenotype and reduces the spheroid growth in 3D, which is reflected in vivo in the reduced formation of metastases in a zebrafish PCa xenograft model. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets and that GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation. Citation Format: Verneri Virtanen, Kreetta Paunu, Antti Kukkula, Saana Niva, Ylva Junila, Mervi Toriseva, Terhi Jokilehto, Sari Mäkelä, Riikka Huhtaniemi, Matti Poutanen, Ilkka Paatero, Maria Sundvall. Glucocorticoid receptor-induced non-muscle caldesmon regulates growth and metastasis in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A024.

The Erk‐Mediated Phosphorylation of Non‐Muscle Caldesmon Alters Osteoclastogenesis in RANKL‐Induced Raw264.7 Cells

The RANKL-RANK signaling pathway is the key event for osteoclastogenesis accompanied by the podosome formation for cell adhesion or extracellular matrix remodeling. Recently, we showed that non-muscle caldesmon (l-CaD) is a key constituent of podosome in RANKL-induced osteoclasts (OCs). Using different decoy peptides mimicking the Pak- or Erk-mediated phosphorylation at the C-terminal end of l-CaD along with full-length mutants of l-CaD also suggested that the l-CaD phosphorylation may involve in the control of osteoclastogenesis. In this study, we used the Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) to examine the altering effects of the Erk signaling pathway leading to changes of l-CaD phosphorylation on the RANKL-induced TRAP activity and cell-cell fusion in osteoclastogenesis. Our study showed that administrations of t-BHQ concentrations of 1, 10, 25, and 50 microM for 30 min or 24 hr before RANKL induction caused increases of l-CaD phosphorylation by 1.13, 1.14, 1.82, and 1.88 folds, respectively, in concomitant with the increased number of small size of differentiated OCs by 2-, 3-, 4-, and 4-folds, respectively, but the decreased fusion index by 0.66, 0.5, 0.3, and 0.3 in a dose dependent manner. However, complete inhibition of Erk phosphorylation with the concomitant 50~60% decrease of l-CaD phosphorylation by treatment with 10 microM PD0325901 for 30 min or 24 hr before RANKL induction completely abolished the OC differentiation. Results obtained here suggest that the Erk-activated l-CaD phosphorylation is required for RANKL-induced OC differentiation, however the dynamic control of l-CaD unphosphorylation/phosphorylation is important for the control of cell-cell fusion in osteoclastogenesis. Support or Funding Information This study is supported by Ministry of Science and Technology of Taiwan Government ( MOST-106-2320-B-005-003-MY2) RANKL-induced formation of actin ring around the cell periphery in differentiated OCs stained with total l-CaD/F-actin (top panel, A), phosphorylated p-l-CaD/F-actin (bottom, A), and vinculin/F-actin (B), and resolved by confocal immunofluorescence imaging. Actin ring structure is composed of the core (labeled with l-CaD, presumbably unphosphorylated) and the peripheral rim (labeled with vinculin, as well as phosphorylated p-l-CaD). Treatments with t-BHQ concentrations of 1, 10, 25, and 50 microM for 24 hr increased l-CaD phosphorylation. Treatments with t-BHQ concentrations of 1, 10, 25, and 50 microM for 24 hr before RANKL induction increased the number of small size of differentiated OCs, but decreased the fusion index in a dose dependent manner. Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) were used for 30 min and 24 hr to examine alterations of Erk and l-CaD phosphorylation. Erk activator (i.e. t-BHQ) and inhibitors (i.e. PD98059 and PD0325901) were used for 30 min and 24 hr to examine the altering effects on RANKL-induced TRAP activity and cell-cell fusion in osteoclastogenesis. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

L-caldesmon alters cell spreading and adhesion force in RANKL-induced osteoclasts

BackgroundOsteoclasts (OCs) are motile multinucleated cells derived from differentiation and fusion of hematopoietic progenitors of the monocyte-macrophage lineage that undergo a multistep process called osteoclastogenesis. The biological function of OCs is to resorb bone matrix for controlling bone strength and integrity, which is essential for bone development. The bone resorption function is based on the remodelling of the actin cytoskeleton into an F-actin-rich structure known as the sealing zone for bone anchoring and matrix degradation. Non-muscle caldesmon (l-CaD) is known to participate in the regulation of actin cytoskeletal remodeling, but its function in osteoclastogenesis remains unclear.Methods/resultsIn this study, gain and loss of the l-CaD level in RAW264.7 murine macrophages followed by RANKL induction was used as an experimental approach to examine the involvement of l-CaD in the control of cell fusion into multinucleated OCs in osteoclastogenesis. In comparison with controls, l-CaD overexpression significantly increased TRAP activity, actin ring structure and mineral substrate resorption in RANKL-induced cells. In contrast, gene silencing against l-CaD decreased the potential for RANKL-induced osteoclastogenesis and mineral substrate resorption. In addition, OC precursor cells with l-CaD overexpression and gene silencing followed by RANKL induction caused 13% increase and 24% decrease, respectively, in cell fusion index. To further understand the mechanistic action of l-CaD in the modulation of OC fusion, atomic force microscopy was used to resolve the mechanical changes of cell spreading and adhesion force in RANKL-induced cells with and without l-CaD overexpression or gene silencing.Conclusionsl-CaD plays a key role in the regulation of actin cytoskeletal remodeling for the formation of actin ring structure at the cell periphery, which may in turn alter the mechanical property of cell-spreading and cell surface adhesion force, thereby facilitating cell-cell fusion into multinucleated OCs during osteoclastogenesis.

Effect of l-caldesmon on osteoclastogenesis in RANKL-induced RAW264.7 cells.

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation.

Role of myocyte enhancing factor 2B in epithelial myofibroblast transition of human gingival keratinocytes

It has recently emerged that the myogenic contribution of the epithelial mesenchymal transition plays a role in neoplastic invasion and metastasis. Myocyte enhancing factor 2B (MEF2B) is the only MEF2 isoform expressed during early embryonic development, and is herein proposed to transactivate the downstream target proteins of the epithelial myofibroblast transition (EMyT). We have previously generated eight preneoplastic cell lines with spindle and cobblestone morphology from human gingival mucosal keratinocytes immortalized by E6/E7 of human papillomavirus type 16. Spindle cells formed tubulogenic morphogenesis on Matrigel and exhibited contractility, anchorage-independent growth and invasiveness to a greater extent than cobblestone cells. Expression of MEF2B mRNA and myofibroblast proteins was higher in spindle cells compared with cobblestone cells. Epidermal growth factor (EGF) treatment of cobblestone cells also induced expression of these genes. Knockdown of MEF2B in a cobblestone cell line abolished EGF-induced upregulation of MEF2, vimentin and non-muscle caldesmon proteins, but enhanced basal expression of mesenchymal vimentin and fibronectin. Differential regulation of intermediate filaments revealed an unrecognized role of MEF2B in myogenic transformation of the epithelial to a myofibroblast phenotype, which occurs as epithelioid variants in some soft tissue sarcomas.

Caldesmon regulates actin dynamics to influence cranial neural crest migration inXenopus

Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca(2+)-calmodulin-binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration.

E0708 IGFBP-7 contributes to vascular remolding during hypertension development in ouabain-induced hypertensive rats

PurposesThe aim of this study was to elucidate the potential new targets for vascular remolding with hypertension.Methods and ResultsIn this study, we established the ouabain-induced hypertensive SD rats (OHR), the...

Abstract 5660: Essential Role of Non-muscle Myosin Light Chain Kinase in Hypoxia-induced Murine Pulmonary Hypertension

Introduction: The pulmonary arterial hypertension (PAH) phenotype shares several pathways involved in cancer pathobiology. We previously demonstrated that the multi-kinase inhibitor, sorafenib, ameliorates rodent PAH (Moreno-Vinasco et al 2008) with genomic signatures which strongly implicated the endothelial cell cytoskeleton in the development of PAH vascular remodeling including non-muscle caldesmon, Rho-kinase and both smooth muscle- and non-muscle myosin light chain kinase (nmMLCK) isoforms. We hypothesized that the acto-myosin machinery present within lung endothelium are critical to the development of PAH in a murine model of hypoxia-induced pulmonary hypertension. Methods: Wild type (WT) and genetically engineered mice with targeted deletion of the nmMLCK allele were exposed to normoxia, or to hypoxia and SU5416, a VEGFR-1,2 inhibitor that induces severe PH (3.5 weeks). WT and nmMLCK KO mice were assessed for hemodynamics and histopathology. Results: Hypoxia-SU5416 WT mice developed pulmonary artery phenotype with significant increases in diphosphorylated MLC levels (2-fold, no change in total MLC), increased mean right ventricular systolic pressures (RVSP) and hypertrophy (RV/LV+S) and increased hematocrit when compared to WT-normoxic controls. However, in response to hypoxia/SU, nmMLCK-KO mice displayed reduced pMLC, significant decreases in right ventricular hypertrophy (27.3% decrease), in RVSP (25% decrease) without changes in hematocrit. Histopathology indicates reduced peri-vascular infiltration and remodeling in the nmMLCK KO mice compared to WT controls. Conclusions: Our results are consistent with the notion that myosin light chains (MLC) are a canonical and downstream mediator of mechanical events underlying the actin-myosin interactions in lung endothelium during PH remodeling and nmMLCK is a potential therapeutic target in PAH-mediated remodeling.

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 transgene expression disrupts focal adhesions in HTM cells and increases outflow facility in organ-cultured human and monkey anterior segments

Cytoskeleton modulating compounds have been shown to lower intraocular pressure (IOP) and increase outflow facility. Caldesmon is one protein that is involved in the regulation of actin stress fiber formation. The effects of rat non-muscle caldesmon (Cald) gene over-expression on focal adhesions in human trabecular meshwork (HTM) cells and on outflow facility in organ-cultured human and monkey anterior segments were determined. Treatment of HTM cells with adenovirus-delivered caldesmon (AdCaldGFP) resulted in characteristic changes in the actin cytoskeleton and matrix adhesions within 24–48 hr post-transduction. Stress fibers gradually disappeared and novel actin structures were formed (see manuscript by Grosheva et al., this issue). In cells with disrupted stress fibers, vinculin-containing focal adhesions were also disrupted. In organ-cultured anterior segments, baseline outflow facility (μl min −1 mmHg −1) for all anterior segments averaged (mean± sem): human, 0.19±0.03 ( n=12); monkey, 0.36±0.02 ( n=19). In human anterior segments, transduction with 10 7 plaque forming units of AdGFPCald increased outflow facility by 43±21% ( p≤0.11, n=6) at 66 hr compared to baseline and corrected for the changes in outflow facility of the contralateral vehicle treated segment. Using the same time point, i.e. 2–3 days after injection, outflow facility in monkey anterior segments, transduced with 1.5×10 7 plaque forming units of AdGFPCald was increased by 35±18%, p<0.2, n=10 compared to baseline and corrected for the change in outflow facility in the contralateral AdGFP treated segment. Combining human (66 hr) and monkey (2–3 days) data, outflow facility was increased by 38±13%, p<0.02, n=16. Additional analysis of maximum responses in monkey anterior segments from 1 to 6 days after transduction showed outflow facility was increased by 66±18%, p<0.01, n=10. Caldesmon over-expression, which relaxes cultured HTM cells and disrupts their actin cytoskeleton and cell–matrix adhesions, also appears to increase outflow facility in organ-cultured human and monkey anterior segments. This suggests that over-expression of the caldesmon gene in the TM may be an effective approach for the gene therapy of glaucoma.

Role of the Basic C-Terminal Half of Caldesmon in Its Regulation of F-Actin: Comparison between Caldesmon and Calponin

We previously reported that caldesmon (CaD), together with tropomyosin (TM), effectively protects actin filaments from gelsolin, an actin-severing protein. To elucidate the structure/function relationship of CaD, we dissected the functional domain of CaD required for the protection. The basic C-terminal half of rat nonmuscle CaD (D3) inhibits gelsolin activity to the same degree as intact CaD, although a smaller C-terminal region of D3 does not. This smaller C-terminal region contains the minimum regulatory domain responsible for the inhibition of actomyosin ATPase, and for the binding to actin, calmodulin and TM. These results suggest that the domain responsible for the inhibition of gelsolin activity lies outside the minimum regulatory domain, and that the positive charge possessed by the C-terminal half of CaD is important for its interaction with actin. Moreover, while the D3 fragment promotes the aggregation of F-actin into bundles as reported previously, this bundle formation is inhibited by the acidic N-terminal half of CaD, as well as by poly-l-glutamate. It seems likely that the acidic N-terminal half of CaD neutralizes the superfluous basic feature of the C-terminal half. A comparison between D3 and calponin, another actin-binding protein that is also basic and has similar actin-regulatory activities, is also discussed.

Nonmuscle caldesmon: its distribution and involvement in various cellular processes

Smooth muscle caldesmon is a thin-filament constituent which takes part in the Ca2+-dependent regulation of actomyosin motor activity which converts chemical energy of ATP into force. The molecular anatomy of its counterpart found in a variety of nonmuscle cells is similar. Both contain about 20 nm long terminal domains responsible for functionally important multisite interactions with filamentous actin, tropomyosin, Ca2+/calmodulin, and myosin and differ by a 35 nm long central, alpha-helical fragment which is lacking in nonmuscle caldesmon. The different structural organisation of nonmuscle cells and thus distinct distribution of caldesmon implicates its different physiological functions. Due to direct interaction with globular and filamentous actin as well as with tropomyosin, nonmuscle caldesmon is involved in the assembly, dynamics, or stability of microfilaments, whereas the indirect inhibitory effect on interaction of the microfilaments with myosin causes its participation in the regulation of cell contraction and intracellular motional processes. These functions of nonmuscle caldesmon of vertebrates are controlled by Ca2+/calmodulin (or other Ca2+-binding proteins) or caldesmon phosphorylation catalysed by various protein kinases. Examples of nonmuscle caldesmon involvement in functions of higher and lower eukaryote, animal and plant cells are presented.

Smooth Muscle Hypertrophy Following Partial Bladder Outlet Obstruction Is Associated with Overexpression of Non-Muscle Caldesmon

Partial bladder outlet obstruction (PBOO) induces remodeling of urinary bladder smooth muscle (detrusor). We demonstrate an increase in bladder wall mass, muscle bundle size, and a threefold increase in the cross-sectional area of detrusor myocytes following PBOO in male New Zealand White rabbits compared to that of controls. Some bladders with detrusor hypertrophy function close to normal (compensated), whereas others were dysfunctional (decompensated), showing high intravesical pressure, large residual urine volume, and voiding difficulty. We analyzed the expression of smooth muscle-specific caldesmon (h-CaD) and non-muscle (l-CaD) by Western blotting, RT-PCR, and real-time PCR. The expression of l-CaD is increased significantly at the mRNA and protein levels in the decompensated bladders compared to that of normal and compensated bladders. The CaD was also co-localized with myosin containing cytoplasmic fibrils in cells dissociated from obstructed bladders and cultured overnight. Our data show that the inability of decompensated bladders to empty, despite detrusor hypertrophy, is associated with an overexpression of l-CaD. The level of l-CaD overexpression might be a useful marker to estimate the degree of detrusor remodeling and contractile dysfunction in PBOO.

Activation of p38 MAP-kinase and caldesmon phosphorylation are essential for urokinase-induced human smooth muscle cell migration.

We have explored intracellular pathways involved in the urokinase type plasminogen activator (urokinase or uPA)-stimulated migration of human airway smooth muscle cells (hAWSMC). Using a set of uPA mutants we found that protease activity, growth factor-like and kringle domains of uPA differentially contribute to activation of p42/p44erk1,2 and p38 MAP-kinases. Consistent with our earlier data [Mukhina et al., J. Biol. Chem. 275 (2000), 16450-16458], the kringle domain of uPA was sufficient and required to stimulate cell motility. Here we report that uPA mutants containing the kringle domain specifically activate the p38 MAP-kinase pathway and actomyosin by increasing phosphorylation of the critical Ser-19 on the myosin regulatory light chain and MAP-kinase sites of the actin-associated regulatory protein caldesmon. While pharmacological inhibition of p38 MAP-kinase activation did not affect myosin light chain phosphorylation, it blocked the increase in caldesmon phosphorylation and uPA-stimulated migration of hAWSMC on a collagen-coated surface. We conclude that activation of p38 MAP-kinase and downstream phosphorylation of non-muscle caldesmon is essential for urokinase-stimulated smooth muscle cell migration.

Macrophage caldesmon is an actin bundling protein.

A rapid purification procedure was developed for the isolation of caldesmon (CaD) from rabbit alveolar macrophage. The purified protein migrated with an apparent M(r) of 74,000 +/- 4000 on SDS-PAGE and cross-reacted with anti-gizzard CaD antibodies. A higher M(r) isoform was isolated from chicken gizzard. Their actin-binding parameters and effects on actomyosin-ATPase activity were investigated under identical experimental conditions. Electron microscope studies revealed that macrophage CaD was able to cross-link actin filaments into both networks and bundles. Compact F-actin bundles were predominantly or exclusively seen at cross-linker to actin molar ratios in the 1:20 to 1:10 range. Apparent K(a) at extrapolated saturation of the CaD-binding sites on F-actin was 1.2 x 10(6) M(-1) for macrophage CaD and 1.6 x 10(6) M(-1) for chicken gizzard CaD. CaD from either source was able to stimulate the actin-activated ATPase activity of macrophage myosin. Unexpectedly, chicken gizzard CaD also increased the ATPase activity of gizzard myosin. The degree of stimulation was approximately doubled in the presence of a large excess of Ca(2+)-calmodulin but was unaffected by the presence of macrophage tropomyosin. However, macrophage CaD did not behave as a Ca(2+)- and calmodulin-regulated actin-binding protein. These results, together with published data on other well-characterized actin bundling proteins, suggest that nonmuscle CaD could be essentially involved in the formation and organization of actin bundles at adhesion sites and cell surface projections. However, they afforded no evidence that the macrophage isoform might play a specific role in the Ca(2+)-dependent regulation of actin and myosin II interactions.

Expression of Stress-Response and Cell Proliferation Genes in Renal Cell Carcinoma Induced by Oxidative Stress

Ferric nitrilotriacetate induces oxidative damage in renal proximal tubules that ultimately leads to a high incidence of renal cell carcinoma (RCC) in rats. In search of genes specifically involved in oxystress-induced carcinogenesis, we have applied a modified fluorescent differential display technique to the tumors and an established cell line as well as their non-neoplastic counterparts. We screened approximately 84,000 products. Reverse Northern blotting confirmed differential expression of 20 transcripts, which showed either significant increase, decrease or lack of expression in the RCCs. Five cDNA clones encoded novel products of unknown function. Fifteen cDNA clones were identified by homology search, which included annexin II, Y-box binding protein, ribosomal proteins, heat shock proteins, DNA polymerase, nonmuscle caldesmon (increased); protein tyrosine phosphatase (decreased); selenoprotein P, stromal cell-derived factor 1, intestinal trefoil protein, nicotinamide adenine dinucleotide, reduced form (NADH) dehydrogenase, and insulin-like growth factor binding protein 7 (deleted). Most of the identified genes were associated with stress-response or cellular proliferation. These results suggest that multiple, interactive genetic pathways are involved in carcinogenesis induced by oxidative stress.

Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Caldesmon

Caldesmon is a protein that is found in smooth muscle and in non-muscle cells. Two isoform classes produced by alternative splicing of one gene have been characterized. The smooth muscle, high molecular weight (89–93 kDa), caldesmon isoforms are found in non-muscle and in de-differentiated smooth muscle cells. The conserved regions of all isoforms contain caldesmon's properties such as binding to actin, tropomyosin, Ca 2+-calmodulin, myosin and phospholipids. All isoforms are also very potent inhibitors of the actin-tropomyosin activated myosin MgATPase. Non-muscle and smooth muscle isoforms of caldesmon perform different roles in vivo. This may be reflected by the distinct cellular distribution of these isoform classes. Non-muscle caldesmon is a regulatory factor in the microfilament network and is thus involved in the assembly and stabilization of microfilaments. Smooth muscle caldesmon together with tropomyosin is a mediating factor for Ca 2+-dependent inhibition of smooth muscle contraction.

Alternatively spliced exons of the beta tropomyosin gene exhibit different affinities for F-actin and effects with nonmuscle caldesmon.

The rat beta-tropomyosin (TM) gene expresses two isoforms via alternative RNA splicing, namely skeletal muscle beta-TM and fibroblast TM-1. The latter is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. Skeletal muscle beta-TM contains exons 7 and 10, whereas exons 6 and 11 are used in fibroblasts and smooth muscle. In order to study the properties of the alternatively spliced proteins, recombinant TMs derived from bacterial and insect cell expression systems were produced, including the normal beta gene products, fibroblast TM-1 and beta skeletal muscle TM, two carboxy-terminal chimeric TMs, TM-6/10 and TM-7/11, as well as a carboxyl-truncated version of each, TM-6Cla and TM-7Cla. The purified TM isoforms were used in actin filament association studies. The apparent TM association constants (Ka) were taken as the free concentration at half saturation and were found to be 6 microM for beta Sk TM, 8.5 for TM-6/10, 25 microM for TM-1, and 30 microM for TM-7/11 at an F-actin concentration of 42 microM. For the truncated TMs, the values determined were higher still but the binding was not carried out to full saturation. Isoforms were also produced using the baculovirus-insect cell system which produces proteins with an acetylated amino terminus as is normally found in vivo. This modification significantly enhanced the F-actin association of TM-1 but not the beta skeletal TM or the other isoforms. Fibroblast TM-2 or TM-3, both products of the alpha gene, enhanced the affinity of TM-1 for F-actin, demonstrating different isoforms can act cooperatively on binding to actin. This effect was not detected with the other expressed beta gene products. The presence of 83 kDa nonmuscle caldesmon was found to enhance the binding of TM-1 for F-actin. This effect was dependent on the presence of both exons 6 and 11, as caldesmon had little effect on the other beta gene products. Collectively these results demonstrate TMs differ in their affinity for F-actin, which can be altered by other TMs or actin-binding proteins. The beta tropomyosin isoforms were fluorescently-tagged and microinjected into cultured cells to study their in vivo localization where it was found that each of the full-length TMs bound to microfilaments but, at the light microscopy level, the isoforms were not differentially localized in these fibroblasts.