Gut Microbiota-Mediated Bile Acid Transformations Alter the Cellular Response to Multidrug Resistant Transporter Substrates in Vitro: Focus on P-glycoprotein.

Pharmacokinetic research at the host-microbe interface has been primarily directed toward effects on drug metabolism, with fewer investigations considering the absorption process. We previously demonstrated that the transcriptional expression of genes encoding intestinal transporters involved in lipid translocation are altered in germ-free and conventionalized mice possessing distinct bile acid signatures. It was consequently hypothesized that microbial bile acid metabolism, which is the deconjugation and dehydroxylation of the bile acid steroid nucleus by gut bacteria, may impact upon drug transporter expression and/or activity and potentially alter drug disposition. Using a panel of three human intestinal cell lines (Caco-2, T84, and HT-29) that differ in basal transporter expression level, bile acid conjugation-, and hydroxylation-status was shown to influence the transcription of genes encoding several major influx and efflux transporter proteins. We further investigated if these effects on transporter mRNA would translate to altered drug disposition and activity. The results demonstrated that the conjugation and hydroxylation status of the bile acid steroid nucleus can influence the cellular response to multidrug resistance (MDR) substrates, a finding that did not directly correlate with directionality of gene or protein expression. In particular, we noted that the cytotoxicity of cyclosporine A was significantly augmented in the presence of the unconjugated bile acids deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) in P-gp positive cell lines, as compared to their taurine/glycine-conjugated counterparts, implicating P-gp in the molecular response. Overall this work identifies a novel mechanism by which gut microbial metabolites may influence drug accumulation and suggests a potential role for the microbial bile acid-deconjugating enzyme bile salt hydrolase (BSH) in ameliorating multidrug resistance through the generation of bile acid species with the capacity to access and inhibit P-gp ATPase. The physicochemical property of nonionization is suggested to underpin the preferential ability of unconjugated bile acids to attenuate the efflux of P-gp substrates and to sensitize tumorigenic cells to cytotoxic therapeutics in vitro. This work provides new impetus to investigate whether perturbation of the gut microbiota, and thereby the bile acid component of the intestinal metabolome, could alter drug pharmacokinetics in vivo. These findings may additionally contribute to the development of less toxic P-gp modulators, which could overcome MDR.

Unconjugated Bile Acids Influence Expression of Circadian Genes: A Potential Mechanism for Microbe-Host Crosstalk.

Disruptions to circadian rhythm in mice and humans have been associated with an increased risk of obesity and metabolic syndrome. The gut microbiota is known to be essential for the maintenance of circadian rhythm in the host suggesting a role for microbe-host interactions in the regulation of the peripheral circadian clock. Previous work suggested a role for gut bacterial bile salt hydrolase (BSH) activity in the regulation of host circadian gene expression. Here we demonstrate that unconjugated bile acids, known to be generated through the BSH activity of the gut microbiota, are potentially chronobiological regulators of host circadian gene expression. We utilised a synchronised Caco-2 epithelial colorectal cell model and demonstrated that unconjugated bile acids, but not the equivalent tauro-conjugated bile salts, enhance the expression levels of genes involved in circadian rhythm. In addition oral administration of mice with unconjugated bile acids significantly altered expression levels of circadian clock genes in the ileum and colon as well as the liver with significant changes to expression of hepatic regulators of circadian rhythm (including Dbp) and associated genes (Per2, Per3 and Cry2). The data demonstrate a potential mechanism for microbe-host crosstalk that significantly impacts upon host circadian gene expression.

A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Identification of a Klf4-dependent upstream repressor region mediating transcriptional regulation of the myocardin gene in human smooth muscle cells.

Phenotypic switching of smooth muscle cells (SMCs) plays a central role in the development of vascular diseases such as atherosclerosis and restenosis. However, the factors regulating expression of the human myocardin (Myocd) gene, the master gene regulator of SMC differentiation, have yet to be identified. In this study, we sought to identify the critical factors regulating Myocd expression in human SMCs. Using deletion/genetic reporter analyses, an upstream repressor region (URR) was localised within the Myocd promoter, herein termed PrmM. Bioinformatic analysis revealed three evolutionary conserved Klf4 sites within the URR and disruption of those elements led to substantial increases in PrmM-directed gene expression. Furthermore, ectopic expression established that Klf4 significantly decreased Myocd mRNA levels and PrmM-directed gene expression while electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP) assays confirmed specific binding of endogenous Klf4, and not Klf5 or Klf2, to the URR of PrmM. Platelet-derived growth factor BB (PDGF-BB), a potent inhibitor of SMC differentiation, reduced Myocd mRNA levels and PrmM-directed gene expression in SMCs. A PDGF-BB-responsive region (PRR) was also identified within PrmM, overlapping with the previously identified URR, where either siRNA knockdown of Klf4 or the combined disruption of the Klf4 elements completely abolished PDGF-BB-mediated repression of PrmM-directed gene expression in SMCs. Moreover, ChIP analysis established that PDGF-BB-induced repression of Myocd gene expression is most likely regulated by enhanced binding of Klf4 and Klf5 to a lesser extent, to the PRR of PrmM. Taken together, these data provide critical insights into the transcriptional regulation of the Myocd gene in vascular SMCs, including during SMC differentiation.