ChIP-Seq analysis of transcription factors CREB and C/EBP beta in vasopressin-responsive mouse renal collecting duct mpkCCD cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE98076 | GEO | 2018/03/26
REPOSITORIES: GEO
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Project description:In mammals, the peptide hormone vasopressin controls renal water excretion, largely through regulation of the molecular water channel aquaporin-2 (AQP2) in the renal collecting duct. Regulatory mechanisms of AQP2 show: 1) short-term regulation by membrane trafficking; and 2) long-term regulation involving vasopressin-induced changes in the abundance of the aquaporin-2 protein. Vasopressin activates a G protein-coupled receptor (V2R) increasing cyclic AMP and activating protein kinase PKA. Crebbp and Ep300 are known targets of PKA. They are histone acetyltransferases that acetylate histone H3 lysine-27, a histone mark associated with open chromatin and increased transcription (Tie F et al. Development 2009). The translocation of CREBBP and Ep300 into the nucleus in response to vasopressin in the collecting duct cells, predicts that vasopressin, working through PKA, may increase histone H3K27 acetylation of some genes. We tested this by performing ChIP-Seq for this modification.
2018-01-02 | GSE95007 | GEO
Project description:Vasopressin/cAMP/protein kinase A (PKA) signaling phosphorylates AQP2 water channels in renal collecting ducts to reabsorb water from urine for the prevention of further water loss. Lipopolysaccharide-responsive and beige-like anchor protein (LRBA) mediates vasopressin-induced AQP2 phosphorylation; therefore LRBA is essential for urinary concentration. LRBA is identified as the PKA substrates in a mouse cortical collecting duct principal cell line (mpkCCDcl4) whose phosphorylation levels are nearly perfectly correlated with those of AQP2. Although mouse LRBA contains several consensus PKA phosphorylation sites, their phosphorylation status in response to vasopressin remain unknown. Post-translational modification analysis revealed that RRDS1607 and RRIS2189 were phosphorylated by vasopressin.
2022-07-23 | PXD033859 | Pride
Project description:Vasopressin/cAMP/protein kinase A (PKA)/aquaporin-2 (AQP2) water channels signaling in the kidneys is a canonical pathway that determines AQP2 activity in response to body fluid balance. AQP2 phosphorylation by PKA increases water reabsorption from urine for the prevention of further water loss. We used several activators of cAMP/PKA signaling as screening tools to generate various phosphorylation patterns of PKA substrates and AQP2 in a mouse cortical collecting duct principal cell line (mpkCCDcl4). We next quantified phosphorylation levels of PKA substrates and AQP2 after western blot analysis using a phospho-PKA substrates antibody. Finally, we screen for PKA substrates whose phosphorylation levels were well correlated with those of AQP2. Immunoprecipitation with a phospho-PKA substrates antibody followed by mass spectrometry analysis revealed that the leading candidate in this assay proved to be a lipopolysaccharide-responsive and beige-like anchor protein (LRBA). Phosphorylation levels of LRBA was nearly perfectly correlated with those of AQP2.
2022-07-23 | PXD033852 | Pride
Project description:Vasopressin regulates renal water excretion by binding to the Gs-coupled vasopressin receptor (V2R) in collecting duct cells, resulting in cyclic AMP-dependent increases in epithelial water permeability through regulation of the aquaporin-2 (AQP2) water channel. Our prior studies showed that CRISPR-mediated deletion of protein kinase A (PKA) in cultured mpkCCD cells largely eliminates these regulatory events. These PKA-null cells provide a means of identifying PKA-independent signaling downstream from the V2 receptor. We carried out large-scale quantitative protein mass spectrometry (SILAC) to identify PKA-independent phosphorylation changes in response to V2R-selective vasopressin analog, dDAVP. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Target motif analysis of the phosphopeptides increased in response to dDAVP in PKA-null cells indicates that the vasopressin activates of one or more members of the AMPK/SNF1 subfamily of basophilic protein kinases. Among the upregulated phosphorylation sites were three known targets of SNF1-subfamily kinases, namely Lipe (S559), Crtc1 (S151) and Arhgef2 (S151). One of the phosphorylation sites that increased in occupancy in PKA-null cells was Ser256 of AQP2, a site critical for vasopressin-mediated trafficking of AQP2 to the cell surface. Beyond this, PKA-independent active site phosphorylation changes were also seen for protein kinases Stk39 (SPAK) and Prkci (Protein kinase C iota). Cyclic AMP levels were ~10-fold higher in PKA-null than in PKA-intact cells in the presence of phosphodiesterase inhibitor IBMX, consistent with a marked acceleration of cAMP production in PKA-null cells. The findings are indicative of substantial PKA-independent signaling downstream from the Gs-coupled V2 receptor.
2020-03-05 | PXD015719 | Pride
Project description:Vasopressin, a peptide hormone, controls renal water excretion, largely through regulation of water channel aquaporin-2 (AQP2) in the renal collecting duct. There are two regulatory mechanisms of AQP2: 1) short-term regulation by membrane trafficking of AQP2; and 2) long-term regulation involving vasopressin-induced changes of protein abundance of AQP2 through regulation of gene transcription and protein half-life. Vasopressin binds a G protein-coupled receptor (V2R) activating several downstream signaling pathways. At downstream of V2R activation, many of transcription factors involve gene transcription process associated with status of chromatin structure. ATAC-Seq (Assay for Transpoase-Accessible Chromatin using Sequencing) is a recent technique to study chromatin accessibility (Buenrostro et al. Nat Methods 2013). We carried out ATAC-Seq following standard an ATAC-Seq protocol in mpkCCD cells treated with vehicle or dDAVP for 30 minutes.
2018-03-26 | GSE108786 | GEO
Project description:Background: The transcriptional coactivator with a PDZ-binding motif (TAZ), a downstream effector of the Hippo signaling pathway, regulates the expression of target genes by acting as a transcription cofactor. TAZ knockout (KO) mice exhibit polycystic kidneys and polyuria. This study aimed to investigate the role of TAZ in vasopressin-induced AQP2 regulation. Methods: 1) TAZ knockdown mediated by siRNA in mpkCCDc11 cells; 2) qRT-PCR, semiquantitative immunoblotting, and immunocytochemistry of AQP2; and 3) Next Generation Sequencing (NGS) in mpkCCDc11 cells, a mouse collecting duct cell line. Results: Endogenous expression of AQP2 was induced in mpkCCDc11 cells by treatment with dDAVP (10-9 M) treatment. Treatment with dDAVP (10-9 M) for 24 h increased AQP2 mRNA (12,608 ± 177% of control) and AQP2 protein levels (287 ± 15%). On the contrary, the increase induced by dDAVP in AQP2 mRNA (9,240 ± 241% of the control) and protein levels (215 ± 23%) was significantly reduced in TAZ-KD cells. TonEBP protein levels remained unchanged in TAZ-KD cells. NGS identified several potential AQP2 transcription factors (TF), including Klf6, Irf3, Cebpb, and Nr4a1, selected based on previous in silico database analysis. Among these, Nr4a1 was chosen for further investigation due to its significantly reduced expression of mRNA in TAZ-KD cells, as confirmed by qRT-PCR. Conclusion: TAZ appears to affect dDAVP-induced AQP2 trafficking through mechanisms not mediated by the cAMP/PKA pathway, suggesting the involvement of other non-canonical pathways. TAZ may also regulate AQP2 abundance, potentially through interaction with several transcription factors, including Nr4a1.
2024-09-30 | GSE274937 | GEO
Project description:Purpose: PKA plays a crucial role in vasopressin signaling of renal collecting duct cells. To understand regulation of mRNA expression mediated by vasopressin/PKA signaling, mRNA expression was profiled by RNA-Seq in double knockout cells (both PKA catalytic genes) generated from mouse cortical collecting duct mpkCCD cell line versus control lines with intact PKA expression. Methods: PKA double knockout (dKO) cell lines were generated from mouse cortical collecting duct mpkCCDc11 cells by CRISPR/Cas-9 genome editing method. For mRNA profiling using RNA-Seq analysis, three biological replicates of control (not mutated in PKA two catalytic subunits) cell lines and PKA double knockout cell lines were used. The reads uniquely mapped on GENCODE mouse gene set were analyzed with HOMER (v4.8) and edgeR (v3.10.5). Results and conclusion: About 40-50 million sequence reads per sample were sucessfully mapped in the mouse genome (GENCODE, GPCm38.p5). Among total transcripts of the mouse genome, 10,190 transcripts (cutoff: Counts Per Million > 4 by edgeR) were considered as genes expressed in the cell lines. In differential expression analysis by standard edgeR analysis, 354 transcripts were differentially expressed between control cell lines and PKA dKO cell lines (FDR < 0.05). We also identified nine genes that were markedly decreased in PKA dKO cell lines (log2 PKA dKO/Control < -2, FDR < 0.05) including aquaporin-2 (Aqp2) and two genes that were markedly increased in PKA dKO cell lines (log2 PKA dKO/Control > 2, FDR < 0.05). These results suggest PKA signaling is important for regulation of expression of a very limited number of genes in vasopressin-responsive renal collecting duct cells.
2018-01-02 | GSE95008 | GEO
Project description:Water permeability of the kidney collecting ducts is regulated in part by the amount of the molecular water channel protein aquaporin-2 (AQP2), whose expression, in turn, is regulated by the pituitary peptide hormone vasopressin. We previously showed that stable glucocorticoid receptor knockdown diminished the vasopressin-induced Aqp2 gene expression in the collecting duct cell model mpkCCD. Here, we investigated the pathways regulated by the glucocorticoid receptor by comparing transcriptomes of the mpkCCD cells with or without stable glucocorticoid receptor knockdown. Glucocorticoid receptor knockdown downregulated 5,394 transcripts associated with 55 KEGG pathways including “vasopressin-regulated water reabsorption,” indicative of positive regulatory roles of these pathways in the vasopressin-induced Aqp2 gene expression. Quantitative RT-PCR confirmed the downregulation of the vasopressin V2 receptor transcript upon glucocorticoid receptor knockdown. Glucocorticoid receptor knockdown upregulated 3,785 transcripts associated with 42 KEGG pathways including the “TNF signaling pathway” and “TGFβ signaling pathway,” suggesting the negative regulatory roles of these pathways in the vasopressin-induced Aqp2 gene expression. Quantitative RT-PCR confirmed the upregulation of TNF and TGFβ receptor transcripts upon glucocorticoid receptor knockdown. TNF or TGFβ inhibitor alone, in the absence of vasopressin, did not induce Aqp2 gene transcription. However, TNF or TGFβ blunted the vasopressin-induced Aqp2 gene expression. In particular, TGFβ reduced vasopressin-induced increases in Akt phosphorylation without inducing epithelial-to-mesenchymal transition or interfering with vasopressin-induced apical AQP2 trafficking. In summary, our RNA-seq transcriptomic comparison revealed positive and negative regulatory pathways maintained by the glucocorticoid receptor for the vasopressin-induced Aqp2 gene expression.
2022-05-16 | GSE194044 | GEO
Project description:Prior studies have implicated myosin light chain kinase (MLCK) in the regulation of aqua-porin-2 (AQP2) in the renal collecting duct. To discover signaling targets of MLCK, we deleted the Mylk- gene to obtain MLCK-null mpkCCD cells and carried out comprehensive phosphopro-teomics using the SILAC method for quantification. Only 107 phosphopeptides were changed in abundance by MLCK deletion out of 1743 quantified over multiple replicates (29 decreased and 78 increased). One of the decreased phosphopeptides corresponded to the canonical target site in myosin regulatory light chain (MLC). Network analysis indicated that targeted phosphopro-teins clustered into distinct structural/functional groups: “acto-myosin,” “signaling,” “nuclear envelope,” “gene transcription,” “mRNA processing,” “energy metabolism,” “intermediate fila-ments,” “adherens junctions” and “tight junctions.” There was significant overlap between the derived MLCK signaling network and a previously determined PKA signaling network. Phalloidin labeling revealed that MLCK deletion inhibited the normal effect of vasopressin to depolymer-ize F-actin. Immunocytochemistry and electron microscopy demonstrated a defect in pro-cessing of early endosomes to late endosomes. In contrast, surface biotinylation studies showed no effect of MLCK deletion on AQP2 endocytosis. We conclude that MLCK is part of a multi-component signaling pathway that includes much more than simple regulation of conven-tional non-muscle myosins through MLC phosphorylation. Furthermore, vasopressin signaling in collecting duct cells involves both MLCK-dependent signaling and PKA-dependent signaling, which display significant overlap. Mapping MLCK targets revealed potential roles in both nu-cleus and cytoplasm. The latter includes proteins that regulate both actin polymerization and AQP2-endosomal processing from early to late endosomes.
2020-03-04 | PXD014985 | Pride
Project description:Vasopressin is the major hormone that regulates renal water excretion. It does so by binding to a receptor in renal collecting duct cells, triggering signaling pathways that ultimately regulate the abundance, location, and activity of the water channel protein aquaporin 2. We took an advantage of quantitative large scale proteomic technologies and oligonucleotide microarrays to quantify steady state changes in protein and transcript abundances in response to vasopressin in a collecting duct cell line, mpkCCD clone 11 (Yu et al. PNAS 2009, 106:2441-2446). This cell line originally developed by Alan Vandewalle’s group recapitulates vasopressin-mediated AQP2 expression and phosphorylation as seen in native colleting duct cells.
2010-12-09 | GSE19938 | GEO