Xiaoyang Mo

CXXC5 Associates with Smads to Mediate TNF-α Induced Apoptosis.

Apoptosis is a widespread phenomenon and its dysregulation may result in a variety of human pathologies, such as cancer, autoimmune diseases and neurodegenerative disorders. CXXC-type zinc finger protein 5 (CXXC5) is commonly considered as a tumor suppressor undergoing deregulation or deletion in hematonosis. But it has implied involvement in apoptosis indirectly and the molecular mechanism remains unknown. In this study, we investigated CXXC5-induced apoptosis as well as its underlying mechanism. A fluorescence resonance energy transfer (FRET) assay suggested that CXXC5 induced cell death and caspase-3 activity in primary rat cortical neurons. Further colorimetric TUNEL assay, Hoechst staining and flow cytometric assay indicated a time-dependent apoptosis in which the activities of caspase-8 and caspase-3 were both regulated via CXXC5 according to enzymatic activity assay, Hoechst staining and Western blotting. Transcription reporter assay and Western blotting showed that CXXC5 resulted in activation of tumor necrosis factor-α (TNF-α), initiated the extrinsic apoptosis pathway and cross-linked with the intrinsic mitochondrial pathway. Being a bone morphogenetic protein 4 (BMP-4) downstream regulator, and also a transcription factor, cellular co-localization and co-immunoprecipitation results indicate that CXXC5 co-localized and interacted with Smads. Western blotting and nuclear fraction extraction implied that CXXC5 facilitated Smad3 phosphorylation and Smad4 nuclear translocation, and that co-expression of Smad together with CXXC5 resulted in higher TNF-α reporter activity. In sum, CXXC5 appears to regulate the TNF-α apoptosis pathway by associating with Smads.

SMYD1, an SRF-Interacting Partner, Is Involved in Angiogenesis

Previous studies have demonstrated that Smyd1 plays a critical role in cardiomyocyte differentiation, cardiac morphogenesis and myofibril organization. In this study, we uncovered a novel function of Smyd1 in the regulation of endothelial cells (ECs). Our data showed that Smyd1 is expressed in vascular endothelial cells, and knockdown of SMYD1 in endothelial cells impairs EC migration and tube formation. Furthermore, Co-IP and GST pull-down assays demonstrated that SMYD1 is associated with the Serum Response Factor (SRF). EMSA assays further showed that SMYD1 forms a complex with SRF and enhances SRF DNA binding activity. Our studies indicate that SMYD1 serves as an SRF-interacting protein, enhances SRF DNA binding activity, and is required for EC migration and tube formation to regulate angiogenesis.

CXXC5 regulates differentiation of C2C12 myoblasts into myocytes

CXXC5 is a member of the CXXC-type zinc-finger domain containing protein family, which is suggested to function in gene transcription, cell adhesion and cytoskeleton organization. Previous studies have revealed that CXXC5 is expressed in skeletal muscle, but whether it regulates skeletal myogenesis is yet unknown. Here, we screened for the possible signaling pathways in which CXXC5 might participate using luciferase gene reporters. The results indicated that CXXC5 significantly increased the activities of the promoters of genes involved in skeletal muscle differentiation. We therefore studied the role of CXXC5 during skeletal myogenesis in C2C12 myoblasts. Our findings suggest that overexpression of CXXC5 in C2C12 myoblasts facilitated myocyte differentiation, while RNAi interference of CXXC5 significantly inhibited the differentiation of C2C12 myoblasts. This study suggests that CXXC5 plays a significant role in regulating skeletal myogenesis.

CXXC5 is required for cardiac looping relating to TGFβ signaling pathway in zebrafish

BACKGROUND CXXC-type zinc-finger protein CXXC5 has been reported to be associated with the development of cardiovascular disease. Recently, through signaling pathway screening we found that CXXC5 activated Tgfβ and myocardial differentiation signaling pathways simultaneously. Although the physiological and pathological function of CXXC5 in many organs has been well elucidated, its function in heart remains unclear. METHODS AND RESULTS Here, we found that zebrafish CXXC5 and SMAD were interacting through ZF-CXXC and MH1 domain. Over-expression of CXXC5 in cardiomyocyte increased the luciferase report activity of Tgfβ signaling pathway. Spatiotemporal expression profile of cxxc5 showed that it consistently expressed during cardiogenesis. Knockdown of cxxc5 in zebrafish displayed looping defects, cardiac dysplasia, pericardial edema, and decreased contraction ability, accompanied with down-regulation of members referring to cardiac looping downstream genes of Tgfβ signaling pathway, such as nkx2.5, hand2, and has2. Co-injection of hand2 mRNA with cxxc5 morpholino rescued the cardiac looping detects. CONCLUSION Our study is the first to provide an in vivo evidence for cxxc5 regulating heart development and cardiac looping via Tgfβ related signaling pathway. This finding suggested that CXXC5 may serve as a possible marker that has potential diagnostic and prognostic value in fetus with congenital heart disease.

Microarray profiling analysis uncovers common molecular mechanisms of rubella virus, human cytomegalovirus, and herpes simplex virus type 2 infections in ECV304 cells.

To study the common molecular mechanisms of various viruses infections that might result in congential cardiovascular diseases in perinatal period, changes in mRNA expression levels of ECV304 cells infected by rubella virus (RUBV), human cytomegalovirus (HCMV), and herpes simplex virus type 2 (HSV-2) were analyzed using a microarray system representing 18,716 human genes. 99 genes were found to exhibit differential expression (80 up-regulated and 19 down-regulated). Biological process analysis showed that 33 signaling pathways including 22 genes were relevant significantly to RV, HCMV and HSV-II infections. Of these 33 biological processes, 28 belong to one-gene biological processes and 5 belong to multiple-gene biological processes. Gene annotation indicated that the 5 multiple-gene biological processes including regulation of cell growth, collagen fibril organization, mRNA transport, cell adhesion and regulation of cell shape, and seven down- or up-regulated genes [CRIM1 (cysteine rich transmembrane BMP regulator 1), WISP2 (WNT1 inducible signaling pathway protein 2), COL12A1 (collagen, type XII, alpha 1), COL11A2 (collagen, type XI, alpha 2), CNTN5 (contactin 5), DDR1 (discoidin domain receptor tyrosine kinase 1), VEGF (vascular endothelial growth factor precursor)], are significantly correlated to RUBV, HCMV and HSV-2 infections in ECV304 cells. The results obtained in this study suggested the common molecular mechanisms of viruses infections that might result in congential cardiovascular diseases.

LASS5 Interacts with SDHB and Synergistically Represses p53 and p21 Activity

Longevity Assurance 5 (LASS5), a member of the LASS/Ceramide Synthases family, synthesizes C16-ceramide and is implicated in tumor biology. However, its precise role is not yet well understood. A yeast two-hybrid screen was performed using a human cDNA library to identify potential LASS5- interaction partners. One identified clone encodes succinate dehydrogenase subunit B (SDHB). Mammalian two-hybrid assays showed that LASS5 interacts with SDHB, and the result was also confirmed by GST pull-down and coimmunoprecipitation assays. The C-terminal fragment of SDHB was required for the interaction. LASS5 and SDHB were co-localized in COS-7 cells. LASS5 and SDHB expressions were found to be up-regulated in neuroglioma tissue. Transfection assays showed that LASS5 or SDHB expression repressed p53 or p21 reporter activity, respectively. Simultaneous LASS5 and SDHB expression resulted in stronger repression of p53 and p21 reporter activity, suggesting that LASS5 and SDHB interaction may synergistically affect transcriptional regulation of p53 and p21. Our data provide new molecular insights into potential roles of LASS5 and SDHB in tumor biology.

The LIM protein fhlA is essential for heart chamber development in zebrafish embryos.

Four-and-a-half LIM proteins FHL1-3 play important roles in cardiovascular pathophysiology. However, their roles in heart development remain unclear. Here, we report that fhlA, the zebrafish homolog of human FHL1, was found to be expressed around the 22-somite stage. After 24 hpf, expression was restricted to the heart. fhlA knockdown caused an enlarged cardiac chamber phenotype with up-regulated expression of the cardiac markers, but fhlA overexpression reduced the sizes of the cardiac chambers and down-regulated expression of the markers. The morphology associated with the cmlc2, amhc, and vmhc expression patterns at the 22 somite and 24 hpf stages included a broadened domain in embryos lacking fhlA and a smaller domain in embryos overexpressing fhlA. The changes in the sizes of the chambers were attributed to the changes in the number of ventricular and atrial cells. Loss of fhlA caused a longer heart period and pause between heartbeats in M-modes than in controls, but fhlA overexpression caused shorter systolic and diastolic intervals. Abnormal cardiac chambers and physiological function were found to be largely rescued. We also showed the expression of fhlA in the heart to be increased by retinoic acid (RA) and decreased by the RA synthase inhibitor DEAB. Both fhlA and RA signaling caused a phenotype characterized by the morphological alterations in the chamber sizes, suggesting that the role of fhlA in heart development is probably regulated by RA signaling. Taken together, these results showed that fhlA regulates the size of the heart chamber by reducing the number of cardiac cells.

The bHLH protein Nulp1 is essential for femur development via acting as a cofactor in Wnt signaling in Drosophila.

Background: The basic helix-loop-helix (bHLH) protein families are a large class of transcription factors, which are associated with cell proliferation, tissue differentiation, and other important development processes. We reported that the Nuclear localized protein-1 (Nulp1) might act as a novel bHLH transcriptional factor to mediate cellular functions. However, its role in development in vivo remains unknown. Methods: Nulp1 (dNulp1) mutants are generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal. Expression of Wg target genes are analyzed by qRT-PCR. We use the Top-Flash luciferase reporter assay to response to Wg signaling. Results: Here we show that Drosophila Nulp1 (dNulp1) mutants, generated by CRISPR/Cas9 targeting the Domain of Unknown Function (DUF654) in its C terminal, are partially homozygous lethal and the rare escapers have bent femurs, which are similar to the major manifestation of congenital bent-bone dysplasia in human Stuve-Weidemann syndrome. The fly phenotype can be rescued by dNulp1 over-expression, indicating that dNulp1 is essential for fly femur development and survival. Moreover, dNulp1 overexpression suppresses the notch wing phenotype caused by the overexpression of sgg/GSK3β, an inhibitor of the canonical Wnt cascade. Furthermore, qRT-PCR analyses show that seven target genes positively regulated by Wg signaling pathway are down-regulated in response to dNulp1 knockout, while two negatively regulated Wg targets are up-regulated in dNulp1 mutants. Finally, dNulp1 overexpression significantly activates the Top-Flash Wnt signaling reporter. Conclusion: We conclude that bHLH protein dNulp1 is essential for femur development and survival in Drosophila by acting as a positive cofactor in Wnt/Wingless signaling.

ZNF424 induces apoptosis and inhibits proliferation in lung carcinoma cells.

Background: Previously, we showed that the Zinc finger-containing transcription factor ZNF424 inhibits p21 transcription, which has been widely associated with various cancers. However, because the roles of ZNF424 in tumorigenesis have not been characterized, we correlated ZNF424 expression with tumorigenesis in lung cancer. Results: The present immunohistochemical analyses show significantly lower ZNF424 expression levels in 43 of 60 lung cancer tissues compared with adjacent tissues. Moreover, flow cytometry assays indicated that overexpression of ZNF424 induces apoptosis in A549 human lung carcinoma cells, and overexpression of ZNF424 significantly increases numbers of G1 phase cells and decreases numbers of S phase cells, suggesting that ZNF424 inhibits proliferation. Western Blot analyses show that overexpression of ZNF424 decreases protein expression levels of the mitogen-activated protein kinase (MAPK) signaling proteins P-P38 and P-ERK in A549 cells. Conclusion: These are the first data to associate ZNF424 with tumorigenesis and demonstrate an inhibitory role in lung cancer, indicating the potential of ZNF424 expression as a diagnostic marker of lung tumorigenesis.

Role of Zebrafish fhl1A in Satellite Cell and Skeletal Muscle Development

Background: Four-and-a-half LIM domains protein 1 (FHL1) mutations are associated with human myopathies. However, the function of this protein in skeletal development remains unclear. Methods: Whole-mount in situ hybridization and embryo immunostaining were performed. Results: Zebrafish Fhl1A is the homologue of human FHL1. We showed that fhl1A knockdown causes defective skeletal muscle development, while injection with fhl1A mRNA largely recovered the muscle development in these fhl1A morphants. We also demonstrated that fhl1A knockdown decreases the number of satellite cells. This decrease in satellite cells and the emergence of skeletal muscle abnormalities were associated with alterations in the gene expression of myoD, pax7, mef2ca and skMLCK. We also demonstrated that fhl1A expression and retinoic acid (RA) signalling caused similar skeletal muscle development phenotypes. Moreover, when treated with exogenous RA, endogenous fhl1A expression in skeletal muscles was robust. When treated with DEAB, an RA signalling inhibitor which inhibits the activity of retinaldehyde dehydrogenase, fhl1A was downregulated. Conclusion: fhl1A functions as an activator in regulating the number of satellite cells and in skeletal muscle development. The role of fhl1A in skeletal myogenesis is regulated by RA signaling.