Gregory B. Vanden Heuvel

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.

Dynamic expression of murine Cux2 in craniofacial, limb, urogenital and neuronal primordia

CDP/Cut homeodomain factors are a divergent group of transcriptional repressors that are conserved during metazoan evolution. The cut locus of Drosophila is required for external sensory organ development and dorso-ventral boundary formation in the wing. In vertebrates, two CDP/Cut genes have been identified, Cux1 and Cux2. While Cux1 is widely expressed in mouse embryos particularly in the nephrogenic and urogenital systems, in contrast the early embryonic expression of Cux2 has not been characterized. Here we describe the dynamic profile of Cux2 during mouse embryogenesis. Cux2 expression becomes elaborated in a number of tissues during organogenesis including the olfactory epithelium, branchial arch and limb bud progress zones, roof plate, motor neurons, dorsal root ganglia and urogenital tissues. The conservation of signaling pathways between the branchial arches and limb buds is well documented and surprisingly this can also be applied to the developing urogenital system. Cux2 expression coincides with many tissue regions undergoing proliferative growth or cell rearrangements, however some later expression domains also include sites of programmed cell death. Cux2 is hypothesized to function as a transcriptional regulator that inhibits terminal differentiation and cell cycle exit.

Proximal Tubule Proliferation Is Insufficient to Induce Rapid Cyst Formation after Cilia Disruption

Disrupting the function of cilia in mouse kidneys results in rapid or slow progression of cystic disease depending on whether the animals are juveniles or adults, respectively. Renal injury can also markedly accelerate the renal cyst formation that occurs after disruption of cilia in adult mice. Rates of cell proliferation are markedly higher in juvenile than adult kidneys and increase after renal injury, suggesting that cell proliferation may enhance the development of cysts. Here, we induced cilia loss in the kidneys of adult mice in the presence or absence of a Cux-1 transgene, which maintains cell proliferation. By using this model, we were able to avoid additional factors such as inflammation and dedifferentiation, which associate with renal injury and may also influence the rate of cystogenesis. After induction of cilia loss, cystic disease was not more pronounced in adult mice with the Cux-1 transgene compared with those without the transgene. In conclusion, these data suggest that proliferation is unlikely to be the sole mechanism underlying the rapid cystogenesis observed after injury in mice that lose cilia function in adulthood.

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.

Ectopic expression of Cux1 is associated with reduced p27 expression and increased apoptosis during late stage cyst progression upon inactivation of Pkd1 in collecting ducts

Polycystic kidney diseases (PKD) are inherited disorders characterized by fluid‐filled cysts primarily in the kidneys. We previously reported differences between the expression of Cux1, p21, and p27 in the cpk and Pkd1 null mouse models of PKD. Embryonic lethality of Pkd1 null mice limits its study to early stages of kidney development. Therefore, we examined mice with a collecting duct specific deletion in the Pkd1 gene. Cux1 was ectopically expressed in the cyst lining epithelial cells of newborn, P7 and P15 Pkd1CD mice. Cux1 expression correlated with cell proliferation in early stages of cystogenesis, however, as the disease progressed, fewer cyst lining cells showed increased cell proliferation. Rather, Cux1 expression in late stage cystogenesis was associated with increased apoptosis. Taken together, our results suggest that increased Cux1 expression associated with apoptosis is a common feature of late stage cyst progression in both the cpk and Pkd1CD mouse models of PKD. Developmental Dynamics 240:1493–1501, 2011.

CD14 : a candidate biomarker for the prognosis of polycystic kidney disease

Zhou et al. validate the expression of markers of the innate immune system in the cpk mouse model of polycystic kidney disease (PKD), in human recessive PKD, and in human autosomal dominant PKD and show that CD14 expression correlates with PKD progression, even from very early stages of disease. Moreover, they show that CD14 is expressed from the renal tubule epithelial cells, suggesting a mechanism of Toll-like receptor-4 activation in PKD prior to the infiltration of inflammatory cells.

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.