A rapid degradation of calponin 2 is required for cytokinesis.

Calponin 2 is an actin cytoskeleton-associated protein and plays a role in regulating cell motility-related functions such as phagocytosis, migration, and division. We previously reported that overexpression of calponin 2 inhibits the rate of cell proliferation. To investigate the underlying mechanism, our present study found that the levels of endogenous calponin 2 in NIH3T3 and HEK293 cells rapidly decreased before cell division characterized by an absence at the actin contractile ring. In cells lacking endogenous calponin 2, transfective expression of GFP-fusion calponin 2 inhibited cell proliferation similar to that of nonfusion calponin 2. Fluorescent imaging studies of mitotic cells indicated that a proper level of calponin 2 expression and effective degradation during cytokinesis are necessary for normal cell division. Computer-assisted dynamic image analysis of dividing cells revealed that overexpression of calponin 2 significantly affects motility and shape behaviors of cells only on the interval from the start of anaphase to the start of cytokinesis, i.e., the pre-cytokinesis phase, but not on the interval from the start of cytokinesis to 50% completion of cytokinesis. The pre-cytokinesis degradation of calponin 2 was attenuated by MG132 inhibition of the ubiquitin proteasome and inhibitor of protein kinase C (PKC), suggesting that PKC phosphorylation-triggered degradation of calponin 2 could determine the rate of cytokinesis. The novel role of calponin 2 in regulating the rate of cytokinesis may be targeted for therapeutic applications such as in an inhibition of malignant tumor growth.

Intercalated disc protein Xinß is required for Hippo-YAP signaling in the heart.

Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinß, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinß in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinß mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinß knock-out mice-indicating a functional and genetic interaction between Xinß and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinß modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.

Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms.

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.

Effect of eradication of Helicobacter pylori on the histology and cellular phenotype of gastric intestinal metaplasia.

BACKGROUND & AIMS: Eradication of Helicobacter pylori appears to reduce gastric cancer incidence. We examined the effect of successful H pylori therapy on histology, phenotype of gastric intestinal metaplasia (GIM) (complete vs incomplete), and expression of several biomarkers related to carcinogenesis. METHODS: Ninety-six H pylori-positive patients from Japan were treated successfully and followed up prospectively over 4 years with yearly endoscopy and were classified into 3 groups: group CG, chronic gastritis without GIM (n = 36); group IM, chronic gastritis with GIM (n = 33); group DYS, and GIM with dysplasia/cancer in a different location of the stomach (n = 27). A total of 288 endoscopic procedures were performed. Histology, mucin-histochemistry, and immunoperoxidase assays using monoclonal antibodies (mAbs) for cell phenotype (monoclonal antibody Das-1/colonic) and for neoplasia (TC22 and p53) were performed. RESULTS: The GIM histologic score was higher in group DYS than in group IM (P < .05) and group CG (P < .0001). The GIM scores did not change in groups IM and DYS over 4 years. mAb Das-1 reactivity was higher in group DYS (63%) than in group IM (39%) and group GC (0%). After eradication of H pylori, mAb Das-1 reactivity disappeared in 40% of patients (P < .0001) despite the unchanged GIM scores, and regression of TC22-4 was noted in the same patients. CONCLUSIONS: H pylori eradication does not reduce the histologic GIM score, but changes the cellular phenotype of GIM. This change of phenotype may be an important factor in the reduction of cancer incidence after eradication of H pylori.

Caldesmon mutant defective in Ca(2+)-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility.

Despite intensive in vitro studies, little is known about the regulation of caldesmon (CaD) by Ca(2+)-calmodulin (Ca(2+)-CaM) in vivo. To investigate this regulation, a mutant was generated of the C-terminal fragment of human fibroblast CaD, termed CaD39-AB, in which two crucial tryptophan residues involved in Ca(2+)-CaM binding were each replaced with alanine. The mutation abolished most CaD39-AB binding to Ca(2+)-CaM in vitro but had little effect on in vitro binding to actin filaments and the ability to inhibit actin/tropomyosin-activated heavy meromyosin ATPase. To study the functional consequences of these mutations in vivo, we transfected an expression plasmid carrying CaD39-AB cDNA into Chinese hamster ovary (CHO) cells and isolated several clones expressing various amounts of CaD39-AB. Immunofluorescence microscopy revealed that mutant CaD39-AB was distributed diffusely throughout the cytoplasm but also concentrated at membrane ruffle regions. Stable expression of CaD39-AB in CHO cells disrupted assembly of stress fibers and focal adhesions, altered cell morphology, and slowed cell cycle progression. Moreover, CaD39-AB-expressing cells exhibited motility defects in a wound-healing assay, in both velocity and the persistence of translocation, suggesting a role for CaD regulation by Ca(2+)-CaM in cell migration. Together, these results demonstrate that CaD plays a crucial role in mediating the effects of Ca(2+)-CaM on the dynamics of the actin cytoskeleton during cell migration.

Role of VEGF family members and receptors in coronary vessel formation.

The specific roles of vascular endothelial growth factor (VEGF) family members and their receptors (VEGFRs) in coronary vessel formation were studied. By using the quail heart explant model, we found that neutralizing antibodies to VEGF-B or VEGF-C inhibited tube formation on the collagen gel more than anti-VEGF-A. Soluble VEGFR-1, a receptor for VEGF-A and -B, inhibited tube formation by 87%, a finding consistent with that of VEGF-B inhibition. In contrast, addition of soluble VEGFR-2, a receptor for VEGF family members A, C, D, and E, inhibited tube formation by only 43%. Acidic FGF-induced tube formation dependency on VEGF was demonstrated by the attenuating effect of a soluble VEGFR-1 and -2 chimera. The localization of VEGF R-2 and R-3 was demonstrated by in situ hybridization of serial sections, which documented marked accumulations of transcripts for both receptors at the base of the truncus arteriosus coinciding with the temporal and spatial formation of the coronary arteries by means of ingrowth of capillary plexuses. This finding suggests that both VEGFR-2 and R-3 may play a role in the formation of the coronary artery roots. In summary, these experiments document a role for multiple members of the VEGF family and their receptors in formation of the coronary vascular bed.

Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer.

BACKGROUND & AIMS: Nonmuscle human tropomyosin (hTM) isoforms have distinct functions and may play important roles in various disease processes. METHODS: In an attempt to identify colon epithelial tropomyosin isoform, a complementary DNA library prepared from a human colon cancer cell line T84 was screened by an oligonucleotide probe complementary to messages of all known hTM isoforms. A novel clone called TC22 was obtained. The amino acid sequence of TC22 isoform is identical to isoform 5 (hTM5) apart from the C terminal domain, amino acids 222-247 coding the exon 9. RESULTS: Northern blot analysis showed that TC22 message is expressed in transformed epithelial cell lines and tumor tissues but not in normal epithelial cells. We developed a monoclonal antibody specific to TC22 isoform (TC22-4). By Western blot and immunoperoxidase assays, we analyzed 105 colonic specimens (fresh frozen and formalin fixed) from 96 patients with colon polyps (hyperplastic) or adenomas with or without dysplasia and cancer. Twenty-one of 22 (95%) of colon cancer specimens showed the presence of TC22, compared with only 1 of the 17 normal colon specimens and none of the 13 hyperplastic polyps (P < 0.0001). As assayed by immunoperoxidase staining, TC22 expression progressively increased in benign adenomatous polyps (35%) and polyps with mild and severe dysplasia (57% and 100%, respectively). CONCLUSIONS: We cloned and sequenced a novel hTM isoform, TC22, which is strongly associated with colonic neoplasia and carcinoma. TC22 may provide a useful biomarker for surveillance of colon cancer.