Dual-specificity phosphatase 29 is induced during neurogenic skeletal muscle atrophy and attenuates glucocorticoid receptor activity in muscle cell culture.

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Newly named dual-specificity phosphatase 29 (Dusp29) has been identified as a novel neurogenic atrophy-induced gene in skeletal muscle. Quantitative PCR analysis revealed that Dusp29 expression is significantly higher in differentiated myotubes compared with proliferating myoblasts. To determine how Dusp29 is transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dusp29 were cloned, fused to a reporter gene, and found to be highly inducible in response to ectopic expression of the myogenic regulatory factors (MRF), MyoD and myogenin. Furthermore, site-directed mutagenesis of conserved E-box elements within the proximal promoter of Dusp29 rendered a Dusp29 reporter gene unresponsive to MRF overexpression. Dusp29, an atypical Dusp also known as Dupd1/Dusp27, was found to attenuate the ERK1/2 branch of the MAP kinase signaling pathway in muscle cells and inhibit muscle cell differentiation when ectopically expressed in proliferating myoblasts. Interestingly, Dusp29 was also found to destabilize AMPK protein while simultaneously enriching the phosphorylated pool of AMPK in muscle cells. Additionally, Dusp29 overexpression resulted in a significant increase in the glucocorticoid receptor (GR) protein and elevation in GR phosphorylation. Finally, Dusp29 was found to significantly impair the ability of the glucocorticoid receptor to function as a transcriptional activator in muscle cells treated with dexamethasone. Identifying and characterizing the function of Dusp29 in muscle provides novel insights into the molecular and cellular mechanisms for skeletal muscle atrophy.

Perspectives on implementing mobile health technology for living kidney donor follow-up: In-depth interviews with transplant providers.

BACKGROUND: United States transplant centers are required to report follow-up data for living kidney donors for 2 years post-donation. However, living kidney donor (LKD) follow-up is often incomplete. Mobile health (mHealth) technologies could ease data collection burden but have not yet been explored in this context. METHODS: We conducted semi-structured in-depth interviews with a convenience sample of 21 transplant providers and thought leaders about challenges in LKD follow-up, and the potential role of mHealth in overcoming these challenges. RESULTS: Participants reported challenges conveying the importance of follow-up to LKDs, limited data from international/out-of-town LKDs, and inadequate staffing. They believed the 2-year requirement was insufficient, but expressed difficulty engaging LKDs for even this short time and inadequate resources for longer-term follow-up. Participants believed an mHealth system for post-donation follow-up could benefit LKDs (by simplifying communication/tasks and improving donor engagement) and transplant centers (by streamlining communication and decreasing workforce burden). Concerns included cost, learning curves, security/privacy, patient language/socioeconomic barriers, and older donor comfort with mHealth technology. CONCLUSIONS: Transplant providers felt that mHealth technology could improve LKD follow-up and help centers meet reporting thresholds. However, designing a secure, easy to use, and cost-effective system remains challenging.

Cadherins and catenins in cancer: connecting cancer pathways and tumor microenvironment.

Cadherin-catenin complexes are integral components of the adherens junctions crucial for cell-cell adhesion and tissue homeostasis. Dysregulation of these complexes is linked to cancer development via alteration of cell-autonomous oncogenic signaling pathways and extrinsic tumor microenvironment. Advances in multiomics have uncovered key signaling events in multiple cancer types, creating a need for a better understanding of the crosstalk between cadherin-catenin complexes and oncogenic pathways. In this review, we focus on the biological functions of classical cadherins and associated catenins, describe how their dysregulation influences major cancer pathways, and discuss feedback regulation mechanisms between cadherin complexes and cellular signaling. We discuss evidence of cross regulation in the following contexts: Hippo-Yap/Taz and receptor tyrosine kinase signaling, key pathways involved in cell proliferation and growth; Wnt, Notch, and hedgehog signaling, key developmental pathways involved in human cancer; as well as TGFß and the epithelial-to-mesenchymal transition program, an important process for cancer cell plasticity. Moreover, we briefly explore the role of cadherins and catenins in mechanotransduction and the immune tumor microenvironment.

A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma.

Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic xenografts derived from patients with GBM. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due, at least in part, to increased phosphorylation, cytoplasmic retention, and reduced activity of the YAP/TAZ transcriptional coactivators. Furthermore, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells, irrespective of their inherent response to TMZ. The data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment.

Fam83d modulates MAP kinase and AKT signaling and is induced during neurogenic skeletal muscle atrophy.

Skeletal muscle atrophy is a serious health condition that can arise due to aging, cancer, corticosteroid exposure, and denervation. Previous work comparing gene expression profiles in control and denervated muscle tissue revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR and Western blot analysis found that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Characterization of the transcriptional regulation of Fam83d showed that ectopic expression of myogenic regulatory factors inhibits Fam83d reporter gene activity. To assess where Fam83d is localized in the cell, Fam83d was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize in a punctate manner to the cytoplasm of muscle cells. To assess function, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation, the MAP Kinase signaling pathway, and the AKT signaling pathway were analyzed. Fam83d overexpression resulted in significant repression of myosin heavy chain and myogenin expression, while phosphorylated ERK and AKT were also significantly repressed. Interestingly, inhibition of the 26S proteasome and the MAP kinase signaling pathway both resulted in stabilization of Fam83d during muscle cell differentiation. Finally, Fam83d has a putative phospholipase D-like domain that appears to be necessary for destabilizing casein kinase Iα and inhibiting ERK phosphorylation in cultured myoblasts. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP kinase and AKT signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.

Inhibition of protein phosphatase methylesterase 1 dysregulates MAP kinase signaling and attenuates muscle cell differentiation.

Protein phosphatase methylesterase 1 has been identified as a novel gene in skeletal muscle that is upregulated in response to neurogenic atrophy in mice. Western blot analysis confirms that Ppme1 is expressed during both muscle cell proliferation and differentiation. Additionally, the Ppme1 promoter is active in muscle cells, while mutation of a conserved E-box element prevents full induction of the Ppme1 reporter gene, suggesting that Ppme1 is transcriptionally regulated by myogenic regulatory factors. Interestingly, immunofluorescence analysis indicates that Ppme1 is localized to both the cytoplasm and the nucleus, while cell fractionation shows that Ppme1 is found only in the cytoplasm. Functional studies reveal that inhibition of Ppme1 using ABL127 or AMZ30 attenuates muscle cell differentiation. Interestingly, inhibition of Ppme1 by ABL127 led to a significant increase in AP-1 reporter activity, as well as, increases in ERK1/2, c-Jun, Ppme1, and PP2A protein levels in differentiating muscle cells. In contrast, AMZ30 treated cells showed a significant decrease in AP-1 reporter activity and a decrease in ERK1/2 and p38 phosphorylation levels. Finally, co-immunoprecipitation studies show that ABL127, but not AMZ30, causes disruption of the endogenous interaction between Ppme1 and PP2A. The data in this study show for the first time that Ppme1 is expressed in skeletal muscle and is upregulated in response to neurogenic atrophy. Furthermore, these findings suggest that Ppme1 may act as a sentinel of the MAP kinase signaling pathway and may indirectly regulate the ERK1/2 and p38 branches via a non-canonical mechanism leading to inhibition of muscle cell differentiation.

Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of food and water-borne E. coli-mediated human diarrhoea worldwide. The incidence in developing countries is estimated at 650 million cases per year, resulting in 800 000 deaths, primarily in children under the age of five. ETEC is also the most common cause of diarrhoea among travellers, including the military, from industrialized nations to less developed countries. In addition, ETEC is a major pathogen of animals, being responsible for scours in cattle and neonatal and postweaning diarrhoea in pigs and resulting in significant financial losses. Studies on the pathogenesis of ETEC infections have concentrated on the plasmid-encoded heat-stable and heat-labile enterotoxins and on the plasmid-encoded antigenically variable colonization factors. Relatively little work has been carried out on chromosomally encoded virulence factors. Here, we review the known virulence factors of ETEC and highlight the future for combating this major disease.

The unusual extended signal peptide region of the type V secretion system is phylogenetically restricted.

The plasmid encoded toxin, Pet, is a prototypical member of the serine protease autotransporters of the Enterobacteriaceae. In addition to the passenger and beta-domains typical of autotransporters, in silico predictions indicate that Pet possesses an unusually long N-terminal signal sequence. The signal sequence can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which we have termed the extended signal peptide region, demonstrate remarkable conservation. In contrast, the N2, H2 and C regions demonstrate significant variability and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the extended signal peptide region remains obscure and surprisingly it has not been proven that the extended signal peptide region is actually synthesized as part of the signal sequence. Here, we demonstrate that the extended signal peptide region is present only in Gram-negative bacterial proteins originating from the classes Beta- and Gammaproteobacteria, and more particularly only in proteins secreted via the Type V secretion pathway: autotransporters, TpsA exoproteins of the two-partner system and trimeric autotransporters. In vitro approaches demonstrate that the DNA region encoding the extended signal peptide region is transcribed and translated.

A conserved extended signal peptide region directs posttranslational protein translocation via a novel mechanism.

Members of the type V secretion family are among the most prevalent secreted proteins in Gram-negative bacteria. A subset of this family, including Pet, the prototypical member of the Enterobacteriaceae serine proteases, possess unusual signal peptides which can be divided into five regions termed N1 (charged), H1 (hydrophobic), N2, H2 and C (cleavage site) domains. The N1 and H1 regions, which the authors have named the extended signal peptide region (ESPR), demonstrate remarkable conservation. In contrast, the N2, H2 and C regions show significant variability, and are reminiscent of typical Sec-dependent signal sequences. Despite several investigations, the function of the ESPR remains obscure. Here, it is shown that proteins possessing the ESPR are translocated in a posttranslational fashion. The presence of the ESPR severely impairs inner membrane translocation. Mutational analysis suggests that the ESPR delays inner membrane translocation by adopting a particular conformation, or by interacting with a cytoplasmic or inner membrane co-factor, prior to inner membrane translocation.