Xiushan Wu

Regulation of bone formation and remodeling by G-protein-coupled receptor 48

G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48-/- mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway.

The Rho family of small GTPases: crucial regulators of skeletal myogenesis

Abstract.The Rho family of small GTPases is involved in a diverse array of cellular processes, including regulation of the actin cytoskeleton, cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity. Recent findings have implicated the Rho proteins as key regulators of the skeletal myogenic program; however, much controversy presently exists as to the precise role of these proteins in this process. This review examines the present controversial findings pertaining to the Rho family’s regulation of skeletal myogenesis and extrapolates from both other differentiation systems and recently published data the possible mechanisms by which these proteins function in the myogenic cascade.

SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin

SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.

WDR26: A novel Gβ‐like protein, suppresses MAPK signaling pathway

WD40 repeat proteins play important roles in a variety of cellular functions, including cell growth, proliferation, apoptosis, and intracellular signal transduction. Mitogen‐activated protein kinases (MAPKs) are evolutionary conserved enzymes in cell signal transduction connecting cell‐surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that G‐protein coupled receptors (GPCRs) play important roles in the signal transduction from extracellular stimuli to MAPKs and the WD40‐containing Gβ proteins as well as Gβ‐like proteins are involved in the stimulation and regulation of the MAPK signaling pathways. Here we report the identification and characterization of a novel human WD40 repeat protein, WD40 repeat protein 26 (WDR26). The cDNA of WDR26 is 3,729 bp, encoding a Gβ‐like protein of 514 amino acids in the cytoplasm. The protein is highly conserved in evolution across different species from yeast, Drosophila, mouse, to human. Northern blot analysis indicates that WDR26 is expressed in most of the examined human tissues, especially at a high level in skeletal muscle. Overexpression of WDR26 in the cell inhibits the transcriptional activities of ETS proteins, ELK‐1 and c‐fos serum response element (SRE), mediated by MEKK1. These results suggest that WDR26 may act as a negative regulator in MAPK signaling pathway and play an important role in cell signal transduction.

Neurochemical phenotype of vagal afferent neurons activated to express c-fos in response to luminal stimulation in the rat

UNLABELLED The vagus nerve conveys meal-induced primary afferent responses to the brainstem. Electrophysiological studies indicate that luminal stimuli such as osmolarity and the digestion products of carbohydrates elicit powerful vagal nodose neuronal responses by activating serotonin 3 (5-hydroxytryptamine-3, 5-HT3) receptors on intestinal mucosal afferent fibers. To characterize the neurochemical phenotype of neurotransmitters in vagal nodose neurons that are activated by luminal stimulation, we examined c-fos protein (c-Fos) expression in response to luminal stimulation in conscious rats. A double-labeling technique using antisera to glutamate (Glu), substance P (SP), calcitonin gene-related peptide (CGRP), and somatostatin (SS) was used to determine the neurochemical profile of c-Fos-positive neurons. c-Fos immunoreactivity was insignificant in vehicle-treated rats. Luminal perfusions of NaCl (500 mOsm), tap water (5 mOsm), maltose (300 mmol/l), and 5-HT (10(-5) mol/l) each elicited a significant increase in the number of cells expressing c-Fos. Chronic vagotomy eliminated an increase in nodose neuronal c-Fos expression, and the 5-HT3 receptor antagonist granisetron significantly reduced it. Glu-, SP-, and CGRP-containing neurons represented 28%, 53%, and 19%, respectively, of the total population of nodose neurons. Few neurons contained SS. Double-labeling studies revealed that of the c-Fos-positive neurons responsive to hypertonic NaCl, 52%, 41%, and 3% exhibited immunoreactivity for Glu, SP, and CGRP, respectively. Of those responsive to tap water, 47%, 50%, and 4% exhibited immunoreactivity for Glu-, SP- and CGRP, respectively. In addition, 44%, 38%, and 8% of 5-HT-stimulated and 30%, 32%, and 5% of maltose-stimulated c-Fos-positive neurons exhibited, respectively, Glu, SP, and CGRP immunoreactivity. The few neurons that contained SS did not express c-Fos. CONCLUSIONS Vagal primary afferent neurons that respond to 5-HT-dependent luminal stimuli, such as hyperosmolarity and maltose, contain mainly Glu and SP. These neurons appear to play an important role in the mediation of the vago-vagal reflex elicited by luminal stimuli.

Identification and characterization of two novel zinc finger genes, ZNF359 and ZFP28, in human development.

Transcription factors play an essential role in controlling gene expression during cardiac and vascular pathogeneses. Identification of regulatory genes in the cardiovascular system is a necessary step toward an understanding of the pathogenesis of congenital heart disease and acquired cardiovascular diseases. The Cys2/His2 type zinc finger genes are the single largest class of transcription factors in the human genome and many numbers of these krüpple-like zinc finger genes have been found to be involved in cardiac development or cardiovascular diseases. In this study, we have identified two novel human krüpple-like zinc finger genes named ZNF359 and ZFP28 from the human heart cDNA library. The complete human ZNF359 cDNA sequence is 3270bp and contains a 1932-bp open reading frame (ORF) that encodes a 643 amino acid protein with an N-terminal KRAB domain and 16 C-terminus zinc finger C2H2 motifs. The ZFP28 cDNA sequence is 4104bp and contains a 2076-bp ORF that encodes an 868 amino acid protein with an N-terminal signal peptide, two KRAB domains, and 14 C-terminal C2H2 zinc finger motifs. Northern blot analyses showed a strong expression of ZNF359 and ZFP28 in various tissues of adult human. A further analysis using human embryonic tissues (18-23 weeks) showed a development-specific expression pattern in heart, skeletal muscle, liver, lung, kidney, and brain, suggesting a role for these genes in embryonic development.

Functional protease-activated receptors in the dorsal motor nucleus of the vagus

Background  Protease‐activated receptors (PARs), a family member of G‐protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR‐1 and PAR‐2 in the dorsal motor nucleus of the vagus (DMV).

Expression of a novel Krüpple-like zinc-finger gene, ZNF382, in human heart.

With the aim of identifying genes involved in human heart development and disease, we have isolated a novel KRAB-related zinc-finger gene named ZNF382 from heart cDNA library. The ZNF382 gene has a predicted 548-amino acid open reading frame, encoding a putative 64kDa zinc-finger protein. The N-terminus of the ZNF382 coding region has a well-conserved Krüpple-associated box domain that consists of KRAB boxes A and B, whereas the C-terminus contains a Krüpple-type zinc-finger domain possessing nine C(2)H(2) zinc-finger motifs in tandem arrays. The ZNF382 gene is mapped to chromosome 19q13.13. Northern blot analysis indicates that a 2.9-kb transcript specific for ZNF382 is expressed at very early embryonic stage of human (at least earlier than gestation 34 day) and widely in human embryo tissues. At the adult stage, ZNF382 expression is restricted largely to heart tissue suggesting a potential role in heart development and function.

Expression of a novel member of sorting nexin gene family, SNX-L, in human liver development

The sorting nexin (SNX) protein family is implicated in the regulation of receptor degradation and membrane traffic in the cell. With the aim of identifying novel genes involved in receptor degradation and recycling, we have cloned a new member of the sorting nexin gene family, human sorting nexin L, SNX-L (or SNX21). This gene includes 4 exons and 3 introns, and is located on chromosome 20q12-13.1 region, encompassing 8 kb. The full-length cDNA of SNX-L is 1,811 bp, with an open reading frame of 1,092 bp. The protein consists of 364 amino acids and encodes a 40 kDa protein. The SNX-L protein has a common PX domain shared by all SNX family members. The similarity of SNX-L PX domain to the PX consensus sequence is over 40%. PX domains have been shown to associate with specific phospholipids and membrane compartments. Expression analysis of SNX-L mRNA indicates that SNX-L is distinctly and highly expressed in fetus liver, but only weakly expressed in brain, muscle (skeleton muscle, smooth muscle, and cardiac muscle), kidney, and adrenal gland. Strong liver expression of SNX-L is maintained from 12 to 25 weeks during human fetus development, suggesting that SNX-L may be a regulatory gene involved in receptor protein degradation during embryonic liver development.

Caffeic Acid 3,4-Dihydroxy-Phenethyl Ester Induces Cancer Cell Senescence by Suppressing Twist Expression

Compared with traditional cytotoxic cancer therapy, therapy-induced cancer cell senescence attracts much interest because it is similarly effective, has fewer side effects, and is more efficiently cleared by immune cells. In this study, we demonstrate that unlike caffeic acid phenethyl ester, caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE), which is isolated from the medicinal plants Sarcandra glabra and Teucrium pilosum, inhibits human cancer cell growth and colony formation by inducing cancer cell senescence, not apoptosis. CADPE induces cell senescence and morphology changes by increasing cellular size and cytoplasmic granularity, enhancing senescence-associated β-galactosidase activity and differentiated embryo-chondrocyte expressed gene 1 expression, and blocking cell-cycle arrest in the G1 phase. To help understand the underlying mechanisms, we show that CADPE significantly suppressed the expression of Twist1 and led to the up-regulation of rat sarcoma, p53, p21WAF1/CIP1, and p16INK4a proteins in a dose-dependent manner, resulting in the hypophosphorylation of retinoblastoma protein. Furthermore, overexpression of Twist1 prevented CADPE-induced cell senescence in tumor cells. Therefore, our studies provide evidence for a novel role of CADPE in cancer cell senescence by targeting the Twist1-dependent senescence signaling pathway.