Madhulika Sharma

Activation of Notch signaling pathway in HIV-associated nephropathy

Objective:HIV-associated nephropathy (HIVAN) is characterized by the development of glomerulosclerosis and is associated with glomerular epithelial cell proliferation. It has recently been shown that activation of the Notch signaling pathway in podocytes results in glomerulosclerosis and podocyte proliferation. To determine whether Notch signaling is involved in renal disorder associated with HIVAN, we evaluated the expression of Notch receptors in HIVAN. Design:We evaluated the expression of the Notch signaling pathway using an HIV-transgenic (HIV-Tg) rat model of HIVAN, and biopsy samples from HIVAN and normal controls. Methods:Paraffin sections and kidney lysates were used for immunohistochemistry, immunofluorescence and western blot analysis. Results:A collapsing variant of glomerulosclerosis and focal segmental sclerosis was observed in HIV-Tg rats. Glomeruli of HIV-Tg rats demonstrated activation of Notch1 and Notch4, as determined by the presence of the intracellular domains. In addition, we observed increased expression of the Notch target protein, hairy enhancer of split homolog-1 in glomeruli of these animals. The expression of the Groucho homolog transducin-like enhancer protein 4, a Notch effector protein, and the homeodomain protein cut homeobox 1 were also significantly increased in glomeruli of HIV-Tg rats, and this was associated with decreased expression of the cyclin kinase inhibitor p27. Intriguingly, renal biopsy samples from HIVAN patients also showed upregulation of cleaved Notch1 and Notch4 in the glomeruli compared with the expression in normal kidneys. Conclusion:Our results demonstrate activation of Notch signaling pathway in HIVAN, thereby underscoring its role in disease pathogenesis.

Coexpression of Cux-1 and notch signaling pathway components during kidney development

Cux‐1 is the murine homologue of the Drosophila gene cut, which is required for cellular differentiation in several tissues, including the wing margin and Malpighian tubule. Mammalian cut proteins function as cell cycle‐dependent transcriptional repressors in proliferating cells. Targets of Cux‐1 repression include the cyclin kinase inhibitors p21 and p27. However, little is known about the regulation of Cux‐1. In Drosophila, multiple genetic interactions between Cut and the Notch and Wingless signaling pathways occur during wing development. To begin to determine whether Cux‐1 regulation by the Notch signaling pathway is conserved in mammals, we compared the expression patterns of Cux‐1, the murine Notch receptors (Notch 1–4), and the murine ligands (Jagged 1, Jagged 2, and Delta 1) during murine embryogenesis and kidney development. In this report, we demonstrate that Cux‐1 expression overlaps with that of Notch signaling pathway components in multiple tissues during embryonic development. In the developing kidney, Cux‐1 expression overlaps with that of Notch pathway components in the condensing mesenchyme, in pretubular aggregates (comma and S‐shaped bodies), and in the presumptive podocytes of capillary loop stage glomeruli. Furthermore, Cux‐1 was significantly up‐regulated in the rat kidney epithelial cell line RKE expressing a constitutively active Notch 1, and this finding was associated with a reduction of p27. Moreover, Cux‐1 interacts with the Groucho homolog TLE‐4, a corepressor recruited by Notch effector proteins. Taken together, these results suggest that Cux‐1 may function in the context of the Notch signaling pathway in multiple tissues during mammalian embryogenesis. Developmental Dynamics 231:828–838, 2004.

Acceleration of polycystic kidney disease progression in cpk mice carrying a deletion in the homeodomain protein Cux1

Polycystic kidney diseases (PKD) are inherited as autosomal dominant (ADPKD) or autosomal recessive (ARPKD) traits and are characterized by progressive enlargement of renal cysts. Aberrant cell proliferation is a key feature in the progression of PKD. Cux1 is a homeobox gene that is related to Drosophila cut and is the murine homolog of human CDP (CCAAT Displacement Protein). Cux1 represses the cyclin kinase inhibitors p21 and p27, and transgenic mice ectopically expressing Cux1 develop renal hyperplasia. However, Cux1 transgenic mice do not develop PKD. Here, we show that a 246 amino acid deletion in Cux1 accelerates PKD progression in cpk mice. Cystic kidneys isolated from 10-day-old cpk/Cux1 double mutant mice were significantly larger than kidneys from 10-day-old cpk mice. Moreover, renal function was significantly reduced in the Cux1 mutant cpk mice, compared with cpk mice. The mutant Cux1 protein was ectopically expressed in cyst-lining cells, where expression corresponded to increased cell proliferation and apoptosis, and a decrease in expression of the cyclin kinase inhibitors p27 and p21. While the mutant Cux1 protein altered PKD progression, kidneys from mice carrying the mutant Cux1 protein alone were phenotypically normal, suggesting the Cux1 mutation modifies PKD progression in cpk mice. During cell cycle progression, Cux1 is proteolytically processed by a nuclear isoform of the cysteine protease cathepsin-L. Analysis of the deleted sequences reveals that a cathepsin-L processing site in Cux1 is deleted. Moreover, nuclear cathepsin-L is significantly reduced in both human ADPKD cells and in Pkd1 null kidneys, corresponding to increased levels of Cux1 protein in the cystic cells and kidneys. These results suggest a mechanism in which reduced Cux1 processing by cathepsin-L results in the accumulation of Cux1, downregulation of p21/p27, and increased cell proliferation in PKD.

Ouabain activates the Na-K-ATPase signalosome to induce autosomal dominant polycystic kidney disease cell proliferation

The Na-K-ATPase is part of a cell signaling complex, the Na-K-ATPase signalosome, which upon activation by the hormone ouabain regulates the function of different cell types. We previously showed that ouabain induces proliferation of epithelial cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Here, we investigated the signaling pathways responsible for mediating the effects of ouabain in these cells. Incubation of ADPKD cells with ouabain, in concentrations similar to those found in blood, stimulated phosphorylation of the epidermal growth factor receptor (EGFR) and promoted its association to the Na-K-ATPase. In addition, ouabain activated the kinase Src, but not the related kinase Fyn. Tyrphostin AG1478 and PP2, inhibitors of EGFR and Src, respectively, blocked ouabain-dependent ADPKD cell proliferation. Treatment of ADPKD cells with ouabain also caused phosphorylation of the caveolar protein caveolin-1, and disruption of cell caveolae with methyl-β-cyclodextrin prevented Na-K-ATPase-EGFR interaction and ouabain-induced proliferation of the cells. Downstream effects of ouabain in ADPKD cells included activation of B-Raf and MEK and phosphorylation of the extracellular regulated kinase ERK, which translocated into the ADPKD cell nuclei. Finally, ouabain reduced expression of the cyclin-dependent kinase inhibitors p21 and p27, which are suppressors of cell proliferation. Different from ADPKD cells, ouabain showed no significant effect on B-Raf, p21, and p27 in normal human kidney epithelial cells. Altogether, these results identify intracellular pathways of ouabain-dependent Na-K-ATPase-mediated signaling in ADPKD cells, including EGFR-Src-B-Raf-MEK/ERK, and establish novel mechanisms involved in ADPKD cell proliferation.

Hepatomegaly in Transgenic Mice Expressing the Homeobox Gene Cux-1

Cux‐1 is a member of a family of homeobox genes structurally related to Drosophila Cut. Mammalian Cut proteins function as transcriptional repressors of genes specifying terminal differentiation in multiple cell lineages. In addition, mammalian Cut proteins serve as cell‐cycle‐dependent transcriptional factors in proliferating cells, where they function to repress expression of the cyclin kinase inhibitors p21 and p27. Previously we showed that transgenic mice expressing Cux‐1 under control of the CMV immediate early gene promoter develop multiorgan hyperplasia. Here we show that mice constitutively expressing Cux‐1 exhibit hepatomegaly correlating with an increase in cell proliferation. In addition, the increase in Cux‐1 expression in transgenic livers was associated with a decrease in p21, but not p27, expression. Within transgenic livers, Cux‐1 was ectopically expressed in a population of small cells, but not in mature hepatocytes, and many of these small cells expressed markers of proliferation. Transgenic livers showed an increase in α‐smooth muscle actin, indicating activation of hepatic stellate cells, and an increase in cells expressing chromogranin‐A, a marker for hepatocyte precursor cells. Morphological analysis of transgenic livers revealed inflammation, hepatocyte swelling, mixed cell foci, and biliary cell hyperplasia. These results suggest that increased expression of Cux‐1 may play a role in the activation of hepatic stem cells, possibly through the repression of the cyclin kinase inhibitor p21.

The homeodomain protein Cux1 interacts with Grg4 to repress p27kip1 expression during kidney development

The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27(kip1) promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27(kip1) in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27(kip1) at two separate sites in the p27(kip1) promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27(kip1) promoter on the sequence containing two Sp1 sites and a CCAAT box approximately 500 bp from the transcriptional start site, and to an AT rich sequence approximately 1.5 kb from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27(kip1) repression by Cux1 during kidney development.

Inhibition of Notch pathway attenuates the progression of human immunodeficiency virus-associated nephropathy

The Notch pathway is an evolutionarily conserved signaling cascade that is critical in kidney development and has also been shown to play a pathogenetic role in a variety of kidney diseases. We have previously shown that the Notch signaling pathway is activated in human immunodeficiency virus-associated nephropathy (HIVAN) as well as in a rat model of the disease. In this study, we examined Notch signaling in the well established Tg26 mouse model of HIVAN. Notch signaling components were distinctly upregulated in the kidneys of these mice as well as in immortalized podocytes derived from these mice. Notch1 and Notch4 were upregulated in the Tg26 glomeruli, and Notch4 was also expressed in tubules. Notch ligands Jagged1, Jagged2, Delta-like1, and Delta-like 4 were all upregulated in the tubules of Tg26 mice, but glomeruli showed minimal expression of Notch ligands. To examine a potential pathogenetic role for Notch in HIVAN, Tg26 mice were treated with GSIXX, a gamma secretase inhibitor that blocks Notch signaling. Strikingly, GSIXX treatment resulted in significant improvement in both histological kidney injury scores and renal function. GSIXX-treated Tg26 mice also showed diminished podocyte proliferation and dedifferentiation, cellular hallmarks of the disease. Moreover, GSIXX blocked podocyte proliferation in vitro induced by HIV proteins Nef and Tat. These studies suggest that Notch signaling can promote HIVAN progression and that Notch inhibition may be a viable treatment strategy for HIVAN.

Developmental signaling: does it bridge the gap between cilia dysfunction and renal cystogenesis?

For more than a decade, evidence has accumulated linking dysfunction of primary cilia to renal cystogenesis, yet molecular mechanisms remain undefined. The pathogenesis of renal cysts is complex, involving multiple cellular aberrations and signaling pathways. Adding to this complexity, primary cilia exhibit multiple roles in a context-dependent manner. On renal epithelial cells, primary cilia act as mechanosensors and trigger extracellular Ca(2+) influx in response to laminar fluid flow. During mammalian development, primary cilia mediate the Hedgehog (Hh), Wnt, and Notch pathways, which control cell proliferation and differentiation, and tissue morphogenesis. Further, experimental evidence suggests the developmental state of the kidney strongly influences renal cystic disease. Thus, we review evidence for regulation of Ca(2+) and cAMP, key molecules in renal cystogenesis, at the primary cilium, the role of Hh, Wnt, and Notch signaling in renal cystic disease, and the interplay between these developmental pathways and Ca(2+) signaling. Indeed if these developmental pathways influence renal cystogenesis, these may represent novel therapeutic targets that can be integrated into a combination therapy for renal cystic disease.

A possible role of HSP 70 in mediating cardioprotection in patients undergoing CABG

Heat shock protein 70 (HSP70) has been reported to be involved in myocardial self-preservation system. This study shows direct evidence of the effect of HSP70 on lymphocytes during ischemia and reperfusion in CABG (coronary artery bypass graft) surgery. Lymphocytes were separated from the blood obtained from 10 patients undergoing CABG at different time intervals. (i) Baseline samples (drawn before onset of bypass), (ii) ischemic samples (30 min after cross-clamp) and (iii) reperfusion samples (10 min after the cross clamp removal) were incubated with recombinant HSP70 and the cells were harvested after 36 h. The effect of HSP70 was monitored by measuring second messengers such as intracellular calcium, protein kinase C (PKC) and inositol tri phosphate (IP3). In addition CD69 expression was also measured. The results showed a significant decrease in intracellular calcium and CD69 expression in ischemia and further in reperfusion samples as compared to their respective un-triggered controls. PKC and IP3 levels however remained unaffected. The protective effect of HSP70 during ischemia and reperfusion could thus be attributed to decreasing intracellular calcium and CD69 expression. This study could therefore provide a mechanism of cardioprotection afforded by HSP70.

Ectopic expression of the homeobox gene Cux‐1 rescues calcineurin inhibition in mouse embryonic kidney cultures

Cux‐1 is a murine homeobox gene structurally related to Drosophila cut. Cux‐1 is highly expressed in the nephrogenic zone of the developing kidney, where its expression coincides with cell proliferation. Cux‐1 functions as a transcriptional repressor of the cyclin kinase inhibitors (CKI) p21 and p27. Cux‐1 DNA binding activity is negatively regulated by phosphorylation, and dephosphorylation of Cux‐1 results in increased DNA binding. Transgenic mice ectopically expressing Cux‐1 develop renal hyperplasia associated with the down‐regulation of the CKI p27. Calcineurin A (CnA) α (−/−) mice display renal hypoplasia associated with the ectopic expression of p27. CnA is a serine/threonine phosphatase activated by intracellular calcium. Inhibiting CnA with cyclosporin A (CsA) leads to nephron deficit in rat metanephric organ cultures and apoptosis in various renal cell lines. To determine whether the ectopic expression of p27 in CnA‐α −/− kidneys results from the down‐regulation of Cux‐1, metanephroi from embryonic Cux‐1 transgenic and wild‐type mice were harvested and cultured with CsA for 5 days. CsA treatment significantly inhibited growth of wild‐type metanephroi. In contrast, CsA‐treated Cux‐1 transgenic kidney cultures were not growth inhibited, but showed high levels of cell proliferation in the nephrogenic zone. Moreover, in CsA‐treated Cux‐1 transgenic kidney cultures, p27 was not expressed in the nephrogenic zone, but only up‐regulated in maturing glomeruli and tubules. Taken together, our results demonstrate that ectopic expression of Cux‐1 can rescue the effects of CsA inhibition of CnA and suggest that Cux‐1 may be regulated by calcineurin A. Developmental Dynamics 236:184–191, 2007.