CDK8 and CDK19 act redundantly to control the CFTR pathway in the intestinal epithelium
Ontology highlight
ABSTRACT:
SUBMITTER: Dr. Susana Prieto
PROVIDER: S-SCDT-10_15252-EMBR_202154261 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:CDK8 and CDK19 form a conserved cyclin-dependent kinase subfamily that interacts with the essential transcription complex, Mediator, and also phosphorylates the C-terminal domain of RNA polymerase II. Cells lacking either CDK8 or CDK19 are viable and have limited transcriptional alterations, but whether the two kinases redundantly control cell proliferation and differentiation is unknown. Here, we find in mice that CDK8 is dispensable for regulation of gene expression, normal intestinal homeostasis, and efficient tumourigenesis, and is largely redundant with CDK19 in the control of gene expression. Their combined deletion in intestinal organoids reduces long-term proliferative capacity but is not lethal and allows differentiation. However, double-mutant organoids show mucus accumulation and increased secretion by goblet cells, as well as downregulation of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and functionality of the CFTR pathway. Pharmacological inhibition of CDK8/19 kinase activity in organoids and in mice recapitulates several of these phenotypes. Thus, the Mediator kinases are not essential for cell proliferation and differentiation in an adult tissue, but they cooperate to regulate specific transcriptional programmes.
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2019-06-24 | PXD014315 | Pride
Project description:The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G>T (712-1G>T) and c.2657+5G>A (2789+5G>A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657+5G>A; 2562T>G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657+5G>A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT/qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression.
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Project description:BackgroundLoss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function.MethodsOrganoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology.ResultsImmunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure.ConclusionsPro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.
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Project description:Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.
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