Significance of the p38MAPK-CRP2 axis in myofibroblastic phenotypic transition.

We have recently demonstrated that a LIM domain protein, cysteine and glycine-rich protein 2 (CSRP2 [CRP2]), plays a vital role in the functional expression of myofibroblasts and cancer-associated fibroblasts. CRP2 binds directly to myocardin-related transcription factors (MRTF [MRTF-A or MRTF-B]) and serum response factor (SRF) to stabilize the MRTF/SRF/CArG-box complex, leading to the expression of smooth muscle cell (SMC) genes such as α-smooth muscle actin (α-SMA) and collagens. These are the marker genes for myofibroblasts. Here, we show that the adhesion of cultured human skin fibroblasts (HSFs) to collagen reduces the myofibroblastic features. HSF adhesion to collagen suppresses the expression of CRP2 and CSRP2-binding protein (CSRP2BP [CRP2BP]) and reduces the expression of SMC genes. Although CRP2BP is known as an epigenetic factor, we find that CRP2BP also acts as an adaptor protein to enhance the function of CRP2 mentioned above. This CRP2BP function does not depend on its histone acetyltransferase activity. We also addressed the molecular mechanism of the reduced myofibroblastic features of HSFs on collagen. HSF adhesion to collagen inhibits the p38MAPK-mediated pathway, and reducing the p38MAPK activity decreases the expression of CRP2 and SMC genes. Thus, the activation of p38MAPK is critical for the myofibroblastic features. We also show evidence that CRP2 plays a role in the myofibroblastic transition of retinal pigment epithelial cells (RPEs). Like HSFs, such phenotypic modulation of RPEs depends on the p38MAPK pathway.Key words: CRP2, p38MAPK, MRTF, myofibroblasts, retinal pigment epithelial cells.

Role of CRP2-MRTF interaction in functions of myofibroblasts.

Inflammatory response induces phenotypic modulation of fibroblasts into myofibroblasts. Although transforming growth factor-ßs (TGF-ßs) evoke such transition, the details of the mechanism are still unknown. Here, we report that a LIM domain protein, cysteine-and glycine-rich protein 2 (CSRP2 [CRP2]) plays a vital role in the functional expression profile in myofibroblasts and cancer-associated fibroblasts (CAFs). Knock-down of CRP2 severely inhibits the expression of smooth muscle cell (SMC) genes, cell motility, and CAF-mediated collective invasion of epidermoid carcinoma. We elucidate the following molecular bases: CRP2 directly binds to myocardin-related transcription factors (MRTF-A/B [MRTFs]) and serum response factor (SRF) and stabilizes the MRTF/SRF/CArG-box complex to activate SMC gene expression. Furthermore, a three-dimensional structural analysis of CRP2 identifies the amino acids required for the CRP2-MRTF-A interaction. Polar amino acids in the C-terminal half (serine-152, glutamate-154, serine-155, threonine-156, threonine-157, and threonine-159 in human CRP2) are responsible for direct binding to MRTF-A. On the other hand, hydrophobic amino acids outside the consensus sequence of the LIM domain (tryptophan-139, phenylalanine-144, leucine-153, and leucine-158 in human CRP2) play a role in stabilizing the unique structure of the LIM domain.Key words: CRP2, 3D structure, myocardin-related transcription factor, myofibroblast, cancer-associated fibroblasts.

Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma.

Myogenic regulatory factors (MRFs) are pivotal transcription factors in myogenic differentiation. MyoD commits cells to the skeletal muscle lineage by inducing myogenic genes through recruitment of chromatin remodelers to its target loci. This study showed that actin-related protein 5 (Arp5) acts as an inhibitory regulator of MyoD and MyoG by binding to their cysteine-rich (CR) region, which overlaps with the region essential for their epigenetic functions. Arp5 expression was faint in skeletal muscle tissues. Excessive Arp5 in mouse hind limbs caused skeletal muscle fiber atrophy. Further, Arp5 overexpression in myoblasts inhibited myotube formation by diminishing myogenic gene expression, whereas Arp5 depletion augmented myogenic gene expression. Arp5 disturbed MyoD-mediated chromatin remodeling through competition with the three-amino-acid-loop-extension-class homeodomain transcription factors the Pbx1-Meis1 heterodimer for binding to the CR region. This antimyogenic function was independent of the INO80 chromatin remodeling complex, although Arp5 is an important component of that. In rhabdomyosarcoma (RMS) cells, Arp5 expression was significantly higher than in normal myoblasts and skeletal muscle tissue, probably contributing to MyoD and MyoG activity dysregulation. Arp5 depletion in RMS partially restored myogenic properties while inhibiting tumorigenic properties. Thus, Arp5 is a novel modulator of MRFs in skeletal muscle differentiation.

Model-based analysis uncovers mutations altering autophagy selectivity in human cancer.

Autophagy can selectively target protein aggregates, pathogens, and dysfunctional organelles for the lysosomal degradation. Aberrant regulation of autophagy promotes tumorigenesis, while it is far less clear whether and how tumor-specific alterations result in autophagic aberrance. To form a link between aberrant autophagy selectivity and human cancer, we establish a computational pipeline and prioritize 222 potential LIR (LC3-interacting region) motif-associated mutations (LAMs) in 148 proteins. We validate LAMs in multiple proteins including ATG4B, STBD1, EHMT2 and BRAF that impair their interactions with LC3 and autophagy activities. Using a combination of transcriptomic, metabolomic and additional experimental assays, we show that STBD1, a poorly-characterized protein, inhibits tumor growth via modulating glycogen autophagy, while a patient-derived W203C mutation on LIR abolishes its cancer inhibitory function. This work suggests that altered autophagy selectivity is a frequently-used mechanism by cancer cells to survive during various stresses, and provides a framework to discover additional autophagy-related pathways that influence carcinogenesis.

Three-dimensional periodontal tissue regeneration using a bone-ligament complex cell sheet.

Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.

Tumor Progression Is Mediated by Thymosin-ß4 through a TGFß/MRTF Signaling Axis.

Although enhanced thymosin ß4 (TMSB4X/Tß4) expression is associated with tumor progression and metastasis, its tumor-promoting functions remain largely unknown. Here, it is demonstrated that TGFß facilitates Tß4 expression and leads to the activation of myocardin-related transcription factors (MRTF), which are coactivators of serum response factor (SRF) and regulate the expression of genes critical for the epithelial-mesenchymal transition (EMT) and tumor metastasis. In murine mammary gland cells (NMuMG), Tß4 upregulation is required for full induction of a MRTF-regulated EMT gene expression program after TGFß stimulation. Tß4 levels are transcriptionally regulated via the novel cis-acting element AGACAAAG, which interacts with Smad and T-cell factor/lymphoid enhancer factor (TCF/LEF) to synergistically activate the Tß4 promoter downstream of TGFß. Murine skin melanoma cells (B16F0 and B16F1) also show the expression regulation of Tß4 by Smad and TCF/LEF. Tß4-knockout B16F1 (Tß4 KO) clones show significantly diminished expression level of tumor-associated genes, which is regulated by the TGFß/MRTFs pathway. In multiple human cancers, Tß4 levels correlate positively with TGFß1 and the tumor-associated gene expression levels through processes that respectively depend on TGFß receptor 1 (TGFBR1) and MRTF expression. Kaplan-Meier survival analyses demonstrate that high Tß4 expression associates with poor prognosis in an SRF expression-dependent manner in several cancers. In mice, Tß4 KO clones show significantly decreased experimental metastatic potential; furthermore, ectopic expression of constitutively active MRTF-A fully restores the diminished metastatic activity. In conclusion, the TGFß/Tß4/MRTF/SRF pathway is critical for metastasis and tumor progression.Implications: These findings define a molecular mechanism underlying a tumor-promoting function of thymosin ß4 through activation of MRTF/SRF signaling. Mol Cancer Res; 16(5); 880-93. ©2018 AACR.

Stereospecific Inhibitory Effects of CCG-1423 on the Cellular Events Mediated by Myocardin-Related Transcription Factor A.

CCG-1423 suppresses several pathological processes including cancer cell migration, tissue fibrosis, and the development of atherosclerotic lesions. These suppressions are caused by inhibition of myocardin-related transcription factor A (MRTF-A), which is a critical factor for epithelial-mesenchymal transition (EMT). CCG-1423 can therefore be a potent inhibitor for EMT. CCG-1423 and related compounds, CCG-100602 and CCG-203971 possess similar biological activities. Although these compounds are comprised of two stereoisomers, the differences in their biological activities remain to be assessed. To address this issue, we stereoselectively synthesized optically pure isomers of these compounds and validated their biological activities. The S-isomer of CCG-1423 rather than the R-isomer exhibited modestly but significantly higher inhibitory effects on the cellular events triggered by MRTF-A activation including serum response factor-mediated gene expression and cell migration of fibroblasts and B16F10 melanoma cells. Accordingly, the S-isomer of CCG-1423 more potently blocked the serum-induced nuclear import of MRTF-A than the R-isomer. No such difference was observed in cells treated with each of two stereoisomers of CCG-100602 or CCG-203971. We previously reported that the N-terminal basic domain (NB), which functions as a nuclear localization signal of MRTF-A, is a binding site for CCG-1423. Consistent with the biological activities of two stereoisomers of CCG-1423, docking simulation demonstrated that the S-isomer of CCG-1423 was more likely to bind to NB than the R-isomer. This is a first report demonstrating the stereospecific biological activities of CCG-1423.

A novel inhibitory mechanism of MRTF-A/B on the ICAM-1 gene expression in vascular endothelial cells.

The roles of myocardin-related transcription factor A (MRTF-A) and MRTF-B in vascular endothelial cells are not completely understood. Here, we found a novel regulatory mechanism for MRTF-A/B function. MRTF-A/B tend to accumulate in the nucleus in arterial endothelial cells in vivo and human aortic endothelial cells (HAoECs) in vitro. In HAoECs, nuclear localization of MRTF-A/B was not significantly affected by Y27632 or latrunculin B, primarily due to the reduced binding of MRTF-A/B to G-actin and in part, to the low level of MRTF-A phosphorylation by ERK. MRTF-A/B downregulation by serum depletion or transfection of siRNA against MRTF-A and/or MRTF-B induced ICAM-1 expression in HAoECs. It is known that nuclear import of nuclear factor-κB (NF-κB) plays a key role in ICAM-1 gene transcription. However, nuclear accumulation of NF-κB p65 was not observed in MRTF-A/B-depleted HAoECs. Our present findings suggest that MRTF-A/B inhibit ICAM-1 mRNA expression by forming a complex with NF-κB p65 in the nucleus. Conversely, downregulation of MRTF-A/B alleviates this negative regulation without further translocation of NF-κB p65 into the nucleus. These results reveal the novel roles of MRTF-A/B in the homeostasis of vascular endothelium.

RPEL proteins are the molecular targets for CCG-1423, an inhibitor of Rho signaling.

Epithelial-msenchymal transition (EMT) is closely associated with cancer and tissue fibrosis. The nuclear accumulation of myocardin-related transcription factor A (MRTF-A/MAL/MKL1) plays a vital role in EMT. In various cells treated with CCG-1423, a novel inhibitor of Rho signaling, the nuclear accumulation of MRTF-A is inhibited. However, the molecular target of this inhibitor has not yet been identified. In this study, we investigated the mechanism of this effect of CCG-1423. The interaction between MRTF-A and importin α/ß1 was inhibited by CCG-1423, but monomeric G-actin binding to MRTF-A was not inhibited. We coupled Sepharose with CCG-1423 (CCG-1423 Sepharose) to investigate this mechanism. A pull-down assay using CCG-1423 Sepharose revealed the direct binding of CCG-1423 to MRTF-A. Furthermore, we found that the N-terminal basic domain (NB) of MRTF-A, which acts as a functional nuclear localization signal (NLS) of MRTF-A, was the binding site for CCG-1423. G-actin did not bind to CCG-1423 Sepharose, but the interaction between MRTF-A and CCG-1423 Sepharose was reduced in the presence of G-actin. We attribute this result to the high binding affinity of MRTF-A for G-actin and the proximity of NB to G-actin-binding sites (RPEL motifs). Therefore, when MRTF-A forms a complex with G-actin, the binding of CCG-1423 to NB is expected to be blocked. NF-E2 related factor 2, which contains three distinct basic amino acid-rich NLSs, did not bind to CCG-1423 Sepharose, but other RPEL-containing proteins such as MRTF-B, myocardin, and Phactr1 bound to CCG-1423 Sepharose. These results suggest that the specific binding of CCG-1423 to the NLSs of RPEL-containing proteins. Our proposal to explain the inhibitory action of CCG-1423 is as follows: When the G-actin pool is depleted, CCG-1423 binds specifically to the NLS of MRTF-A/B and prevents the interaction between MRTF-A/B and importin α/ß1, resulting in inhibition of the nuclear import of MRTF-A/B.

Prospective randomized controlled trial to compare the effects of omeprazole and famotidine in preventing delayed bleeding and promoting ulcer healing after endoscopic submucosal dissection.

BACKGROUND AND AIMS: Proton pump inhibitors (PPIs) are generally used to prevent delayed bleeding after endoscopic submucosal dissection (ESD) and to heal the artificial ulcers. However, it remains controversial whether PPIs or histamine-2 receptor antagonists (H(2) RAs) are more effective in preventing delayed bleeding after ESD. We prospectively compared the effects of omeprazole and famotidine in preventing delayed bleeding and promoting artificial ulcer healing after ESD. METHODS: A total of 158 patients (155 early gastric cancers and three adenomas) were randomly assigned to the PPI group (omeprazole 20 mg/day) or H(2) RA group (famotidine 40 mg/day) in a prospective randomized controlled trial. The primary end point was the incidence of hematemesis, melena, and/or a decrease in hemoglobin level of 2 g/dL or more requiring endoscopic hemostatic treatment. ESD-induced ulcer healing and changes in ulcer size were also compared at 6 weeks after ESD as a secondary end point. RESULTS: Of the 158 patients, two were excluded from analysis because they had been treated with a PPI before the present study. Accordingly, data from 77 PPI and 79 H(2) RA subjects were included for analysis. Delayed bleeding after ESD occurred in 6.5% of subjects (PPI group) and in 6.3% (H(2) RA group); there was no significant difference between the two groups. Likewise, the two groups were not significantly different with respect to ulcer stage or ulcer size reduction rate. CONCLUSIONS: Proton pump inhibitors are not superior to H(2) RAs for the prevention of delayed bleeding or the healing of artificially induced ulcers after ESD.

Endoscopically treated Cronkhite-Canada syndrome associated with minute intramucosal gastric cancer: an analysis of molecular pathology.

There have been no reports of Cronkhite-Canada syndrome (CCS) associated gastric cancer resected with endoscopy because it is very difficult to identify small cancers that are candidates for endoscopic resection. We report a case of CCS with gastric cancer treated with endoscopic submucosal dissection, and we evaluate the molecular pathological analysis of malignant transformation in patients with CCS. A 74-year-old man had an advanced rectal cancer and gastrointestinal polyposis after presenting with hypoproteinemia, partial hair loss and atrophic nails as well as hyperpigmentation on the hands. He was diagnosed as having CCS. On upper endoscopy, a 7 mm discolored polyp with an irregular microvascular pattern revealed by magnified narrow-band imaging (NBI) was identified in gastric diffuse CCS polyposis. This lesion was treated with endoscopic submucosal dissection and diagnosed as a flat, elevated-type, mucosal well-differentiated tubular adenocarcinoma without lymphatic or venous infiltration, and with tumor-free margins. Microsatellite instability was detected in both the cancer and the surrounding CCS polyps. Mucin-histochemical analysis of the cancer area showed the complete intestinal type, and thus may have differentiated the CCS polyps from that of the common gastric hyperplastic polyps. This case illustrates that a clue to detecting small cancers may be to look for the discolored lesion among reddish CCS polyposis and thereafter to observe the irregular vascular pattern with NBI endoscopy. From the viewpoint of genetic alterations, patients with CCS polyps are considered to be at high risk for developing gastric cancer, and therefore careful follow-up examinations are necessary for the early detection of malignancies.

MRTF-A/B suppress the oncogenic properties of v-ras- and v-src-mediated transformants.

Two members of the myocardin protein family, myocardin-related transcription factor (MRTF)-A and MRTF-B are co-activators of serum response factor (SRF). We recently reported that MRTF-A/B activates the transcription of several actin cytoskeletal/focal adhesion genes SRF dependently, thereby enhancing the formation of stress fibers and focal adhesions. Here, we showed that the levels of caldesmon and tropomyosin, both SRF/MRTF-regulated actin cytoskeletal proteins, were reduced in rat intestinal epithelial (RIE) cell lines that had been transformed with oncogenic ras (RIE-ras) or src (RIE-src) compared with their parental cell line. These cells exhibited morphological abnormalities associated with a disorganized actin cytoskeleton. The serum-stimulated nuclear translocation of MRTF-A/B was suppressed in the RIE-ras and RIE-src cells. However, the transient expression of constitutively active (CA) MRTF-A or MRTF-B reversed the reduced expression levels of caldesmon and tropomyosin and the associated morphological phenotypes. We isolated stable CA-MRTF-A-expressing cell lines from transfected RIE-ras and RIE-src cells and found that their levels of caldesmon and tropomyosin were close to those of untransformed RIE cells. Their morphologies were also normal, with a flattened cell shape and well-developed stress fibers. The CA-MRTF-A-expressing RIE-ras and RIE-src lines also showed lower invasiveness and anchorage-independent growth than their transformed parental cells, in vitro. In vivo, CA-MRTF-A expression suppressed tumor formation and reduced liver metastases. Therefore, we concluded that MRTF-A/B are potent repressors of cancer progression and metastasis and may be good targets for cancer therapy.

Myocardin functions as an effective inducer of growth arrest and differentiation in human uterine leiomyosarcoma cells.

Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. We noticed that the expression of myocardin was markedly downregulated in human uterine leiomyosarcoma cells. Restoration of myocardin expression induced the reexpression of smooth muscle marker proteins and the formation of well-developed actin fibers. A concomitant increase in the expression of a cyclin-dependent kinase inhibitor, p21, led to significantly reduced cell proliferation, via p21's inhibition of the G(1)-S transition. A p21 promoter-reporter assay showed that myocardin markedly increased p21's promoter activity. Furthermore, a serum response factor (SRF)-binding cis-element CArG box in the p21 promoter region was required for this myocardin effect. Chromatin immunoprecipitation and DNA-protein binding assays showed that myocardin indirectly bound to the CArG box in the p21 promoter through the interaction with SRF. Furthermore, immunohistochemistry revealed that the levels of myocardin and p21 were both lower in leiomyosarcoma samples than in normal smooth muscle tissue. Taken together, our results indicate that the downregulation of myocardin expression facilitates cell cycle progression via the reduction of p21 expression in human leimyosarcomas and suggest that myocardin could be a useful therapeutic target for this disease.

[Therapeutic trends of diseases of the upper gastrointestinal tract in patients with negative H. pylori status].

Many diseases of the upper gastrointestinal tract developed in patients with Helicobacter pylori (H. pylori) infection, thus the conditions unrelated to H. pylori are rare. Here, we described the therapeutic trends of diseases in H. pylori negative individuals. Proton pump inhibitor (PPI) is superior to H2 receptor antagonist in both gastroesohageal reflux disease (GERD) including reflux esophagitis and non-erosive reflux disease (NERD) whereas therapeutic gains of PPI treatment for NERD patients are lower than those reported in GERD because of heterogeneity of NERD pathophysiology. Endoscopic therapy for PPI refractory GERD patients remains to be established because there are few studies concerning the effectiveness or safety of the procedures. Main cause of H. pylori-negative ulcer diseases is the use of non-steroidal anti-inflammatory drugs (NSAIDs). PPI therapy is effective for both the prevention and treatment of NSAIDs-induced peptic ulcer. Considerations that should be entertained as causes of intractable peptic ulcers include Zollinger-Ellison syndrome or Crohn's disease. Gastric cancer and carcinoid tumor should be treated with endoscopic or surgical resection regardless of H. pylori infection. As for the treatment for gastric H. pylori-negative MALT lymphoma, radiation therapy (RT) should be selected first, and next chemotherapy will be given to the patients who failed to RT.

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.

Dual roles of myocardin-related transcription factors in epithelial mesenchymal transition via slug induction and actin remodeling.

Epithelial-mesenchymal transition (EMT) is a critical process occurring during embryonic development and in fibrosis and tumor progression. Dissociation of cell-cell contacts and remodeling of the actin cytoskeleton are major events of the EMT. Here, we show that myocardin-related transcription factors (MRTFs; also known as MAL and MKL) are critical mediators of transforming growth factor beta (TGF-beta) 1-induced EMT. In all epithelial cell lines examined here, TGF-beta1 triggers the nuclear translocation of MRTFs. Ectopic expression of constitutive-active MRTF-A induces EMT, whereas dominant-negative MRTF-A or knockdown of MRTF-A and -B prevents the TGF-beta1-induced EMT. MRTFs form complexes with Smad3. Via Smad3, the MRTF-Smad3 complexes bind to a newly identified cis-element GCCG-like motif in the promoter region of Canis familiaris and the human slug gene, which activates slug transcription and thereby dissociation of cell-cell contacts. MRTFs also increase the expression levels of actin cytoskeletal proteins via serum response factor, thereby triggering reorganization of the actin cytoskeleton. Thus, MRTFs are important mediators of TGF-beta1-induced EMT.

Changes in the balance between caldesmon regulated by p21-activated kinases and the Arp2/3 complex govern podosome formation.

Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.

Changes in spatial and temporal localization of Dictyostelium homologues of TRAP1 and GRP94 revealed by immunoelectron microscopy.

TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. In this study, we mainly examined the behavior of Dictyostelium TRAP1 homologue, Dd-TRAP1, during Dictyostelium development by immunoelectron microscopy. In vegetatively growing D. discoideum Ax-2 cells, Dd-TRAP1 locates in nucleolus and vesicles in addition to the cell cortex including cell membrane. Many of Dd-TRAP1 molecules moved to the mitochondrial matrix in response to differentiation, although Dd-TRAP1 on the cell membrane seems to be retained. Some Dd-TRAP1 was also found to be secreted to locate outside the cell membrane in Ax-2 cells starved for 6 h. At the multicellular slug stage, Dd-TRAP1 was primarily located in mitochondria and cell membrane in both prestalk and prespore cells. More importantly, in differentiating prespore cells, a significant number of Dd-TRAP1 locates in the PSV (prespore-specific vacuole) that is a sole cell type-specific organelle and essential for spore wall formation, whereas some Dd-TRAP1 in the cell cortical region of prestalk cells. These findings strongly suggest the importance of Dd-TRAP1 regulated temporally and spatially during Dictyostelium development. Incidentally, we also have certified that the glucose-regulated protein 94 (Dd-GRP94) is predominantly located in Golgi vesicles and cisternae, followed by its colocalization with Dd-TRAP1 in the PSV.