Biomedical subjects

Bradley M Denker

Publications and source records attributed to Bradley M Denker.

9 recordsLinked to original sources

PubMed2006

Cell-cell interactions in the kidney: inducible expression of mutant G protein alpha-subunits in Madin-Darby canine kidney cells for studies of epithelial cell junction structure and function.

Disrupted epithelial cell junctions are a hallmark of numerous disease processes. The signaling mechanisms regulating barrier function and re-establishment of intact junctions after injury and during development are complex and tightly regulated. We have shown that heterotrimeric G proteins regulate assembly and maintenance of epithelial cell barrier in cultured Madin-Darby canine kidney (MDCK) cells. The inducible expression of mutant signaling molecules (constitutively active or dominant negative) in polarized cells is a useful strategy for elucidating the role(s) for specific proteins. Using tetracycline-off inducible expression of wild-type and constitutively active Galpha12, we have demonstrated a fundamental role for Galpha12 in regulating the junction of MDCK cells. Inducible expression permits the comparison of the identical cell line in the presence and absence of the protein of interest and minimizes variation arising from distinct clones. The methods described here are applicable to virtually any protein that may regulate maintenance or assembly of the epithelial cell junctional complex.

Animals↗

PubMed2005

Purkinje cell protein-2 (Pcp2) stimulates differentiation in PC12 cells by Gbetagamma-mediated activation of Ras and p38 MAPK.

Purkinje cell protein-2 (Pcp2 or L7) is highly expressed in cerebellar Purkinje cells and retinal bipolar neurons and interacts with the Galpha(i/o) family of G-proteins. Although the expression pattern of Pcp2 in the developing central nervous system suggests a role in differentiation, its function remains unknown. We established Tet-off inducible expression of Pcp2 in PC12 cells (rat pheochromocytoma cells) to determine whether Pcp2 regulates neuronal differentiation. Utilizing a polyclonal antibody, Pcp2 was localized in the cell body and throughout neurites of differentiated PC12 cells, similar to its localization in cerebellar Purkinje cells. Pcp2 expression in PC12 cells stimulated process formation (5-fold) and NGF (nerve growth factor)-stimulated neurite length (2-fold). Under basal conditions, Pcp2-PC12 cells demonstrated a 5-fold increase in Ras activation relative to non-induced PC12 cells and there was no change in extracellular-signal-regulated kinase 1/2 activity with Pcp2 expression. However, Pcp2 induction led to a >3-fold increase in basal p38 MAPK (mitogen-activated protein kinase) activity and the addition of NGF significantly stimulated both Ras and p38 MAPK in Pcp2-PC12 cells relative to the controls. Pretreatment of Pcp2-PC12 cells with the p38-specific inhibitor SB203580 blocked both the increased neurite formation and NGF-stimulated neurite growth. Pertussis toxin treatment had no effect on neurite growth in control cells, but completely blocked Pcp2-mediated increased neurite growth. Transient transfection of the beta-adrenergic receptor kinase C-terminus to prevent signalling through Gbetagamma in Pcp2-PC12 cells also inhibited the Pcp2-induced phenotype and reduced the Pcp2-stimulated Ras activation. Taken together, these findings demonstrate that Pcp2 induces differentiation in PC12 cells, in part through Gbetagamma-mediated Ras and p38 MAPK activation and suggest the potential for similar signalling mechanisms in Purkinje cells.

Animals↗

PubMed2005

Cultured podocytes establish a size-selective barrier regulated by specific signaling pathways and demonstrate synchronized barrier assembly in a calcium switch model of junction formation.

Podocytes form unique cell-cell junctions (slit diaphragms) that are central to glomerular selectivity, although regulation and mechanisms of slit diaphragm assembly are poorly understood. With the use of cultured podocytes, a paracellular permeability flux assay was established to characterize properties of the size-selective barrier. Paracellular flux of differentiated podocytes was measured using anionic fluorescent dextrans of 3, 10, 40, and 70 kD. Podocytes form a highly selective barrier with a 160-fold difference in flux from the 3-kD dextran (11 pmol/min) to the 70-kD dextran (0.06 pmol/min). Barrier development was dependent on podocyte differentiation and not affected by dextran charge. Puromycin, a known podocyte toxin, increased flux 250% in a dose-dependent manner without affecting cell viability. Screening with modulators of specific signaling pathways identified reversible increases in flux with Src tyrosine and Rho kinase inhibition. The calcium switch model of epithelial junction assembly was modified to determine whether podocytes regulate barrier assembly. When cultured in low calcium for 90 min, flux increased by 300% and consistently returned to baseline 24 to 48 h after switching to normal calcium. Similar to classical epithelial junctions, barrier recovery occurred in the presence of cyclohexamide, an inhibitor of protein synthesis. During the calcium switch, there were reversible changes in localization and detergent solubility of the slit diaphragm protein ZO-1 and alpha-actinin-4, whereas nephrin and podocin solubility were unchanged. Taken together, these findings demonstrate that cultured podocytes develop a selective size barrier that is regulated by specific signaling pathways, and similar to classical epithelial junctions, podocytes demonstrate synchronized assembly of the barrier.

Animals↗

PubMed2004

Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau.

The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons. The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation. Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12). When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QLalpha(12) with tau and the active kinase resulted in 60 +/- 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The Galpha(12) effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.

Actins↗

PubMed2004

Alpha-actinin-4-mediated FSGS: an inherited kidney disease caused by an aggregated and rapidly degraded cytoskeletal protein.

Focal segmental glomerulosclerosis (FSGS) is a common pattern of renal injury, seen as both a primary disorder and as a consequence of underlying insults such as diabetes, HIV infection, and hypertension. Point mutations in the alpha-actinin-4 gene ACTN4 cause an autosomal dominant form of human FSGS. We characterized the biological effect of these mutations by biochemical assays, cell-based studies, and the development of a new mouse model. We found that a fraction of the mutant protein forms large aggregates with a high sedimentation coefficient. Localization of mutant alpha-actinin-4 in transfected and injected cells, as well as in situ glomeruli, showed aggregates of the mutant protein. Video microscopy showed the mutant alpha-actinin-4 to be markedly less dynamic than the wild-type protein. We developed a "knockin" mouse model by replacing Actn4 with a copy of the gene bearing an FSGS-associated point mutation. We used cells from these mice to show increased degradation of mutant alpha-actinin-4, mediated, at least in part, by the ubiquitin-proteasome pathway. We correlate these findings with studies of alpha-actinin-4 expression in human samples. "Knockin" mice with a disease-associated Actn4 mutation develop a phenotype similar to that observed in humans. Comparison of the phenotype in wild-type, heterozygous, and homozygous Actn4 "knockin" and "knockout" mice, together with our in vitro data, suggests that the phenotypes in mice and humans involve both gain-of-function and loss-of-function mechanisms.

Actinin↗

PubMed2004

Polycystin-1L2 is a novel G-protein-binding protein.

Mutations in genes encoding polycystin-1 (PC1) and polycystin-2 cause autosomal dominant polycystic kidney disease. The polycystin protein family is composed of Ca2+-permeable pore-forming subunits and receptor-like integral membrane proteins. Here we describe a novel member of the polycystin-1-like subfamily, polycystin-1L2 (PC1L2), encoded by PKD1L2, which has various alternative splicing forms with two translation initiation sites. PC1L2 short form starts in exon 12 of the long form. The longest open reading frame of PKD1L2 short form, determined from human testis cDNA, encodes a 1775-amino-acid protein and 32 exons, whereas the long form is predicted to encode a 2460-residue protein. Both forms have a small receptor for egg jelly domain, a G-protein-coupled receptor proteolytic site, an LH2/PLAT, and 11 putative transmembrane domains, as well as a number of rhodopsin-like G-protein-coupled receptor signatures. RT-PCR analysis shows that the short form, but not the long form, of human PKD1L2 is expressed in the developing and adult heart and kidney. Furthermore, by GST pull-down assay we observed that PC1L2 and polycystin-1L1 are able to bind to specific G-protein subunits. We also show that PC1 C-terminal cytosolic domain binds to Galpha12, Galphas, and Galphai1, while it weakly interacts with Galphai2. Our results indicate that both PC1-like molecules may act as G-protein-coupled receptors.

Alternative Splicing↗

PubMed2003

Galpha12 regulates epithelial cell junctions through Src tyrosine kinases.

Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that Galpha12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated Galpha12 increases paracellular permeability in Madin-Darby canine kidney (MDCK) cells (Meyer et al. J Biol Chem 277: 24855-24858, 2002). In the present studies, we explore the effects of Galpha12 expression on tight and adherens junction proteins and examine downstream signaling pathways. By confocal microscopy, we detect disrupted tight and adherens junction proteins with increased actin stress fibers in constitutively active Galpha12 (QLalpha12)-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QLalpha12-expressing MDCK cells, consistent with loss of polarity. We found that the tyrosine kinase inhibitor genistein and the Src-specific inhibitor PP-2 reversibly abrogated the QLalpha12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QLalpha12-expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and [3H]mannitol flux was prevented by the inhibitors. Src activity was increased in QLalpha12-expressing MDCK cells as assessed by Src autophosphorylation, and beta-catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that Galpha12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.

Adherens Junctions↗

PubMed2002

Association and colocalization of G protein alpha subunits and Purkinje cell protein 2 (Pcp2) in mammalian cerebellum.

Previously, we have demonstrated a novel interaction between Galpha(o) protein and Purkinje cell protein-2 (Pcp2, also known as L7) in vitro and in transfected cells (Luo and Denker [1999] J. Biol. Chem. 274:10685-10688). Pcp2 is uniquely expressed in cerebellar Purkinje cells and in retinal bipolar neurons, and it may function as a cell-type specific modulator for G protein-mediated cell signaling. This interaction has been further evaluated in the present studies. Coimmunoprecipitation experiments reveal that Pcp2 associates with Galpha(o) in vivo in mouse cerebellum and eye extract. Pcp2 also associate with Galpha(i2) in the cerebellum. No detectable associations of Pcp2 with Galpha(z) and Galpha(q) subunits are observed. The association of Galpha(o) and Pcp2 is detected at postnatal day 1 (P1), and the association remains stable from day 3 (P3) until adulthood. Further, immunofluorescent double labeling and confocal microscopy suggest that Pcp2 and Galpha(o) are colocalized in the distal processes of cerebellar Purkinje cells including axonal endings and dendritic spines. Taken together, these findings indicate colocalization and association of Galpha(o) and Pcp2 in cerebellum and suggest a functional role in regions of synaptic activity.

Age Factors↗

PubMed2002

Zonula occludens-1 is a scaffolding protein for signaling molecules. Galpha(12) directly binds to the Src homology 3 domain and regulates paracellular permeability in epithelial cells.

Zonula occludens proteins are multidomain proteins usually localized at sites of intercellular junctions, yet little is known about their role in regulating junctional properties. Multiple signaling proteins regulate the junctional complex, and several (including G proteins) have been co-localized with zonula occludens-1 (ZO-1) in the tight junction of epithelial cells. However, evidence for direct interactions between signaling proteins and tight junction proteins has been lacking. In these studies, we constructed Galpha-glutathione S-transferase (GST) fusion proteins and tested for interactions with [(35)S]methionine-labeled in vitro translated ZO-1 and ZO-2. Only Galpha(12) directly interacted with in vitro translated ZO-1 and ZO-2. Using a series of ZO-1 domains expressed as GST fusion proteins and in vitro translated [(35)S]methionine-labeled Galpha(12), we found that Galpha(12) and constitutively active (Q229L) alpha(12) (QLalpha(12)) bind to the Src homology 3 (SH3) domain of ZO-1. This binding was not detected with SH3 domains from other proteins. Inducible expression of wild-type alpha(12) and QLalpha(12) in Madin-Darby canine kidney (MDCK) cells was established using the Tet-Off system. In Galpha(12)-expressing cells, we found that ZO-1 and Galpha(12) co-localize by confocal microscopy and co-immunoprecipitate. Galpha(12) from MDCK cell lysates bound to the GST-ZO-1-SH3 domain, and expression of QLalpha(12) in MDCK cells reversibly increased paracellular permeability. These studies indicated that ZO-1 directly interacts with Galpha(12) and that Galpha(12) regulates barrier function of MDCK cells.

Animals↗