1 Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, and 2 Division of Surgery, Children's Hospital, Boston, Massachusetts 02115
Submitted 25 November ...2002
; accepted in final form 8 July 2003
Regulation and assembly of the epithelial cell junctional complex involve multiple signaling mechanisms, including heterotrimeric G proteins. Recently, we demonstrated that G 12 binds to the tight junction scaffolding protein ZO-1 through the SH3 domain and that activated G 12 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 G 12 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 G 12 (QL 12 )-expressing MDCK cells. The normal distribution of ZO-1 and Na-K-ATPase was altered in QL 12 -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 QL 12 phenotype on the junctional complex. Junctional protein localization was preserved in PP-2- or genistein-treated QL 12 -expressing cells, and the increase in paracellular permeability as measured by transepithelial resistance and 3 Hmannitol flux was prevented by the inhibitors. Src activity was increased in QL 12 -expressing MDCK cells as assessed by Src autophosphorylation, and -catenin tyrosine phosphorylation was also increased, although there was no detectable increase in Rho activity. Taken together, these results indicate that G 12 regulates MDCK cell junctions, in part through Src tyrosine kinase pathways.
G proteins; tight junction; adherens junction; Rho
Address for reprint requests and other correspondence: B. M. Denker, Renal Division, Brigham and Women's Hospital and Harvard Medical School, Harvard Institutes of Medicine, 77 Ave. Louis Pasteur, Boston, MA 02115. (E-mail: bdenker{at}rics.bwh.harvard.edu ).
In the present studies, we explore the effects of Galpha12 expression on tight and adherens junction proteins and examine downstream signaling pathways. Taken together, these results indicate that ...G12 regulates Madin-Darby canine kidney (MDCK) cell junctions, in part through Src tyrosine kinase pathways. PUBLICATION ABSTRACT
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)Smethionine-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)Smethionine-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.
In renal tissue injury, activation of the transcription factor NF-kappaB has a central role in the induction of proinflammatory gene expression, which are involved in the development of progressive ...renal inflammatory disease. The function of NF-kappaB during the switch from the inflammatory process toward resolution, however, is largely unknown. Therefore, we assessed the time-dependent activation and function of NF-kappaB in two different models of acute nephritis. Our experiments demonstrate a biphasic activation of NF-kappaB in the anti-Thy-1 model of glomerulonephritis in rats and the LPS-induced nephritis in mice, with a first peak during the induction phase and a second peak during the resolution period. After induction of glomerular immune injury in rats, predominantly NF-kappaB p65/p50 heterodimer complexes are shifted to the nucleus whereas during the resolution phase predominantly p50 homodimers could be demonstrated in the nuclear compartment. In addition, we could demonstrate that p50 protein plays a pivotal role in the resolution of LPS-induced renal inflammation since NF-kappaB p50 knockout mice demonstrate significantly higher chemokine expression, prolonged renal inflammatory cell infiltration with consecutive tissue injury, and reduced survival. In conclusion, our studies indicate that NF-kappaB subunit p50 proteins have critical in vivo functions in immunologically mediated renal disease by downregulating inflammation during the resolution period.
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