PDGF-BB-mediated activation of CREB in vascular smooth muscle cells alters cell cycling via Rb, FoxO1 and p27kip1.

INTRODUCTION & AIM: The vascular response to injury leads to the secretion of several factors, including platelet-derived growth factor (PDGF-BB). PDGF-BB stimulates smooth muscle cell (SMC) conversion to the synthetic phenotype, thereby enhancing proliferation and migration, and contributing to neointimal hyperplasia. Likewise, the cAMP response element binding protein (CREB) transcription factor has been shown to mediate SMC proliferation in response to various mitogens. We therefore investigated the contribution of CREB to PDGF-BB-dependent proliferation of SMCs with the intention of identifying signaling pathways involved both up and downstream of CREB activation. METHODS & RESULTS: Treatments were performed on vascular SMCs from a porcine coronary artery explant model. The role of CREB was examined via adenoviral expression of a dominant-negative CREB mutant (kCREB) as well as inhibition of CREB binding protein (CBP). Involvement of the p27kip1 pathway was determined using a constitutively expressing p27kip1 adenoviral vector. PDGF-BB stimulated transient CREB phosphorylation on Ser-133 via ERK1/2-, PI3-kinase- and Src-dependent pathways. Expression of kCREB decreased PDGF-BB-dependent cell proliferation. PCNA expression and Rb phosphorylation were also inhibited by kCREB. These cell cycle proteins are controlled via p27kip1 expression in response to CREB-dependent post-translational modification of FoxO1. kCREB had no effect on Cyclin D1 expression, but did prevent PDGF-BB-induced Cyclin D1 nuclear translocation. An interaction inhibitor of CBP confirmed that Cyclin D1 is downstream of PDGF-BB and CREB. CONCLUSION: CREB phosphorylation is required for SMC proliferation in response to PDGF-BB. This phenotypic change requires CBP and is mediated by Cyclin D1 and p27kip as a result of changes in FoxO1 activity.

Trans10, cis12 conjugated linoleic acid inhibits 3T3-L1 adipocyte adipogenesis by elevating ß-catenin levels.

BACKGROUND: Trans-10, cis-12 (t10-c12) CLA treatment reduces lipid accumulation in differentiating mouse and human adipocytes, and decreases fat mass in mice, yet the mechanism of action remains unknown. OBJECTIVE: This study investigated the effect of the cis-9, trans-11 (c9-t11) and t10-c12 CLA isomers on the Wnt/ß-catenin pathway, which has been reported to inhibit adipogenesis by down-regulating PPARγ. RESULTS: We observed that t10-c12 CLA treatment of 3T3-L1 adipocytes increases the levels of ß-catenin and Ser-675 phosphorylated ß-catenin due to inhibition of its degradation. These changes in ß-catenin were not linked to either the Wnt/ß-catenin agonist Wnt10b or other upstream effectors such as SFRP-5. Paradoxically, the presence of higher amounts of ß-catenin did not elevate cyclin D1 levels, which is recognized as a critical target gene. Neither of the CLA isomers affected the localization of ß-catenin in the cytosol and nucleus as determined by immunofluorescence microscopy. However, subcellular fractionation suggested the level of cytosolic ß-catenin was reduced in t10-c12 CLA treated cells. Immunoprecipitation revealed that t10-c12 CLA increased the interaction of ß-catenin and PPARγ. CONCLUSIONS: t10-c12-CLA inhibits adipocyte differentiation by increasing ß-catenin stability in 3T3-L1 adipocytes, thus enhancing sequestration of PPARγ in an inactive complex, which prevents progression of adipogenesis.

Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada.

UNLABELLED: Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, ß-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. IMPORTANCE: In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.

Trans-10,cis-12 conjugated linoleic acid (CLA) interferes with lipid droplet accumulation during 3T3-L1 preadipocyte differentiation.

In this study, we hypothesize that the biologically active isomers of conjugated linoleic acid (CLA), cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA, have different effects on early and late stages 3T3-L1 preadipocyte differentiation. Both c9-t11 and t10-c12CLA stimulated early stage pre-adipocyte differentiation (day 2), while t10-c12CLA inhibited late differentiation (day 8) as determined by lipid droplet numbers and both perilipin-1 levels and phosphorylation state. At day 8, the adipokines adiponectin, chemerin and adipsin were all reduced in t10-c12CLA treated cells versus control cells. Immunofluorescence microscopy showed perilipin-1 was present solely on lipid droplets on day 8 in t10-c12 treated 3T3-L1 cells, whereas preilipin-1 was also located in the perinuclear region in control and c9-t11 treated cells. The t10-c12CLA isomer also decreased levels of hormone-sensitive lipase and inhibited lipolysis. These findings indicate that the decrease in lipid droplets caused by t10-c12CLA is the result of an inhibition of lipid droplet production during adipogenesis rather than a stimulation of lipolysis. Additionally, treatment with Gö6976 blocked the effect of t10-c12CLA on perilipin-1 phosphorylation, implicating PKCα in perilipin-1 phosphorylation, and thus a regulator of triglyceride catabolism. These data are supported by evidence that t10-c12CLA activated PKCα. These are the first data to show that CLA isomers can affect lipid droplet dynamics in adipocytes through PKCα.