The broad objectives of the proposed research are to identify the molecular mechanisms underlying the structure, function, and regulation of the inducible nitric oxide synthase (iNOS) genes, and to develop an understanding of their role in renal cell biology. iNOS-generated nitric oxide (NO) plays a critical role in an array of essential functions in many cell types, and it contributes to the control of extracellular volume, and to the pathogenesis of inflammatory and ischemic renal diseases.
In Specific Aim #1, they will characterize the functional properties of the two iNOS isoforms (VSM-NOS and MAC-NOS) expressed in rat kidney, and analyze structure-function correlations. The encoding DNAs of these isoforms will be transfected into tissue cultured cells, and the expressed iNOS isozymes will be functionally analyzed. Chimeric genes and substitution mutants of the isoforms will be constructed and expressed in cultured cells to identify regions of the molecules contributing to distinctive functions. Site-directed, isoform-specific antibodies will be generated for immunolocalization studies.
Specific Aim #2 will explore transcriptional regulation of the VSM-NOS gene under basal conditions and in response to IL-1beta and cAMP in cultured rat glomerular mesangial cells. VSM-NOS promoter-reporter gene constructs, gel shift assays of nuclear extracts, in vitro and in vivo footprinting, and methylation interference assays will be used to characterize the cis-elements and trans-acting factors responsible for transcriptional control of the VSM-NOS gene in these cells.
In Specific Aim #3, they will characterize and compare the molecular mechanisms responsible for iNOS isoform gene regulation during chronic adaptation to changes in salt intake, using in situ hybridization, competitive RT-PCR of microdissected renal structures, immunohistochemistry, immunoblot analysis, and nuclear run-on assays.
Specific Aim #4 will characterize the expression of the iNOS isoforms during the course of renal ischemia-reperfusion injury, using in situ hybridization, immunohistochemistry, and protein and mRNA quantitation. It is anticipated that the results of these studies will provide important insights into the molecular basis for the cell type-specific regulation of the iNOS genes, and their unique roles in renal cell biology and pathobiology. A more complete understanding of NO biosynthesis in the kidney will enable physicians to develop therapeutic strategies designed to enhance the beneficial effects of this molecule, and to minimize its detrimental effects in salt-sensitive hypertension, and inflammatory, immunologic, and ischemic renal diseases.
|Yu, Zhiyuan; Kuncewicz, Teresa; Dubinsky, William P et al. (2006) Nitric oxide-dependent negative feedback of PARP-1 trans-activation of the inducible nitric-oxide synthase gene. J Biol Chem 281:9101-9|
|Yu, Zhiyuan; Kone, Bruce C (2006) Targeted histone H4 acetylation via phosphoinositide 3-kinase- and p70s6-kinase-dependent pathways inhibits iNOS induction in mesangial cells. Am J Physiol Renal Physiol 290:F496-502|
|Jalal, Diane I; Kone, Bruce C (2006) Src activation of NF-kappaB augments IL-1beta-induced nitric oxide production in mesangial cells. J Am Soc Nephrol 17:99-106|
|Yu, Zhiyuan; Xia, Xuefeng; Kone, Bruce C (2005) Expression profile of a human inducible nitric oxide synthase promoter reporter in transgenic mice during endotoxemia. Am J Physiol Renal Physiol 288:F214-20|
|Yu, Zhiyuan; Kone, Bruce C (2004) Hypermethylation of the inducible nitric-oxide synthase gene promoter inhibits its transcription. J Biol Chem 279:46954-61|
|Kuncewicz, Teresa; Sheta, Essam A; Goldknopf, Ira L et al. (2004) Proteomic analysis reveals novel protein targets of S-nitrosylation in mesangial cells. Contrib Nephrol 141:221-30|
|Zhang, Wenzheng; Hayashizaki, Yoshihide; Kone, Bruce C (2004) Structure and regulation of the mDot1 gene, a mouse histone H3 methyltransferase. Biochem J 377:641-51|
|Yu, Zhiyuan; Kone, Bruce C (2004) The STAT3 DNA-binding domain mediates interaction with NF-kappaB p65 and iuducible nitric oxide synthase transrepression in mesangial cells. J Am Soc Nephrol 15:585-91|
|Zou, Lei; Sato, Norio; Kone, Bruce C (2004) Alpha-melanocyte stimulating hormone protects against H2O2-induced inhibition of wound restitution in IEC-6 cells via a Syk kinase- and NF-kappabeta-dependent mechanism. Shock 22:453-9|
|Zhang, Sui; Yang, Yu; Kone, Bruce C et al. (2003) Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/calmodulin-dependent protein kinase II. Circulation 107:1539-44|
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