Myotonic dystrophy protein kinase is critical for nuclear envelope integrity.

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.

The 27-kDa heat shock protein confers cytoprotective effects through a beta 2-adrenergic receptor agonist-initiated complex with beta-arrestin.

Heat shock proteins represent an emerging model for the coordinated, multistep regulation of apoptotic signaling events. Although certain aspects of the biochemistry associated with heat shock protein cytoprotective effects are known, little information is found describing the regulation of heat shock protein responses to harmful stimuli. During screening for noncanonical beta adrenergic receptor signaling pathways in human urothelial cells, using mass spectroscopy techniques, an agonist-dependent interaction with beta-arrestin and the 27-kDa heat shock protein was observed in vitro. Formation of this beta-arrestin/Hsp27 complex in response to the selective beta adrenergic receptor agonist isoproterenol, was subsequently confirmed in situ by immunofluorescent colocalization studies. Radioligand binding techniques characterized a homogeneous population of the beta2 adrenergic receptor subtype expressed on these cells. Using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunoblot analysis and quantitation of caspase-3 activity to detect apoptosis, preincubation of these cells with isoproterenol was found to be sufficient for protection against programmed cell death initiated by staurosporine. RNA interference strategies confirmed the necessity for Hsp27 as well as both beta-arrestin isoforms to confer this cytoprotective consequence of beta adrenergic receptor activation in this cell model. As a result, these studies represent the first description of an agonist-dependent relationship between a small heat shock protein and beta-arrestin to form a previously unknown antiapoptotic "signalosome."

Myotonic dystrophy protein kinase is expressed in embryonic myocytes and is required for myotube formation.

Myotonic dystrophy (DM1) is a multi-systemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). The primary pathophysiology of DM1 is thought to result from RNA transport and processing defects. The function of DMPK in development or any potential role in DM1 remains unknown. Here we report a novel role for DMPK in myogenesis. We have discovered a specific expression pattern of DMPK in mouse and chick embryonic development. DMPK is expressed in postmitotic cardiac and skeletal myocytes and developmental signaling centers. During cardiac myocyte maturation, DMPK migrates from perinuclear to cellular membrane localization. Manipulating DMPK levels in cultured cardiac and skeletal myocytes has revealed a key role for DMPK in myocyte differentiation. Overexpression of DMPK induces cell rounding and apoptosis in myocytes. In addition, DMPK is necessary for myogenin expression in differentiating C2C12 myoblasts.

Conditional expression of Smad7 in pancreatic beta cells disrupts TGF-beta signaling and induces reversible diabetes mellitus.

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-beta (TGF-beta) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-beta signaling, to identify distinct roles for this pathway in adult and embryonic beta cells. Smad7 expression in Pdx1+ embryonic pancreas cells resulted in striking embryonic beta cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1+ cells reduced detectable beta cell expression of MafA, menin, and other factors that regulate beta cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-beta signaling. Thus, our studies reveal that TGF-beta signaling is crucial for establishing and maintaining defining features of mature pancreatic beta cells.

Beta-adrenergic receptor activation in immortalized human urothelial cells stimulates inflammatory responses by PKA-independent mechanisms.

BACKGROUND: Interstitial cystitis (IC) is a debilitating disease characterized by chronic inflammation of the urinary bladder, yet specific cellular mechanisms of inflammation in IC are largely unknown. Multiple lines of evidence suggest that beta-adrenergic receptor (AR) signaling is increased in the inflamed urothelium, however the precise effects of these urothelial cell signals have not been studied. In order to better elucidate the AR signaling mechanisms of inflammation associated with IC, we have examined the effects of beta-AR stimulation in an immortalized human urothelial cell line (UROtsa). For these studies, UROtsa cells were treated with effective concentrations of the selective beta-AR agonist isoproterenol, in the absence or presence of selective inhibitors of protein kinase A (PKA). Cell lysates were analyzed by radioimmunoassay for generation of cAMP or by Western blotting for induction of protein products associated with inflammatory responses. RESULTS: Radioligand binding demonstrated the presence of beta-ARs on human urothelial UROtsa cell membranes. Stimulating UROtsa cells with isoproterenol led to concentration-dependent increases of cAMP production that could be inhibited by pretreatment with a blocking concentration of the selective beta-AR antagonist propranolol. In addition, isoproterenol activation of these same cells led to significant increases in the amount of phosphorylated extracellular signal-regulated kinase (pERK), inducible nitric oxide synthase (iNOS) and the induced form of cyclooxygenase (COX-2) when compared to control. Moreover, preincubation of UROtsa cells with the selective PKA inhibitors H-89 or Rp-cAMPs did not diminish this isoproterenol mediated phosphorylation of ERK or production of iNOS and COX-2. CONCLUSION: Functional beta-ARs expressed on human urothelial UROtsa cell membranes increase the generation of cAMP and production of protein products associated with inflammation when activated by the selective beta-AR agonist isoproterenol. However, the increased production of iNOS and COX-2 by isoproterenol is not blocked when UROtsa cells are preincubated with inhibitors of PKA. Therefore, UROtsa cell beta-AR activation significantly increases the amount of iNOS and COX-2 produced by a PKA-independent mechanism. Consequently, this immortalized human urothelial cell line can be useful in characterizing potential AR signaling mechanisms associated with chronic inflammatory diseases of the bladder.

GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development.

Identification of endogenous signals that regulate expansion and maturation of organ-specific progenitor cells is a major goal in studies of organ development. Here we provide evidence that growth differentiation factor 11 (GDF11), a member of the TGF-beta ligand family, governs the number and maturation of islet progenitor cells in mouse pancreas development. Gdf11 is expressed in embryonic pancreatic epithelium during formation of islet progenitor cells that express neurogenin 3. Mice deficient for Gdf11 harbor increased numbers of NGN3+ cells, revealing that GDF11 negatively regulates production of islet progenitor cells. Despite a marked expansion of these NGN3+ islet progenitors, mice lacking Gdf11 have reduced beta-cell numbers and evidence of arrested beta-cell development, indicating that GDF11 is also required for beta-cell maturation. Similar precursor and islet cell phenotypes are observed in mice deficient for SMAD2, an intracellular signaling factor activated by TGF-beta signals. Our data suggest that Gdf11 and Smad2 regulate islet cell differentiation in parallel to the Notch pathway, which previously has been shown to control development of NGN3+ cells. Thus, our studies reveal mechanisms by which GDF11 regulates the production and maturation of islet progenitor cells in pancreas development.