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Regulation of connexin biosynthesis…

Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b5 and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvaria confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

The myocardium predominantly consists of cardiomyocytes which require strong coupling in order to maintain heart function. Adjacent cardiomyocytes are linked via complex connecting structures, the intercalated discs (ICD). These structures are composed of desmosomes, adherens junctions and gap junctions (Figure 1). Desmosomes, together with adherens junctions, provide cardiomyocyte cohesive strength necessary for strong mechanical coupling. They consist of the transmembrane adhesion molecules Dsg2 and desmocollin 2 (Dsc2), which are linked to the desmin intermediate filament system via the plaque proteins Pg, plakophilin 2 (Pkp2) and desmoplakin (DP) (Figure 1). Mutations affecting the desmosomal components of the ICD can cause Arrythmogenic cardiomyopathy (AC), at least in part because they modulate the functions of the gap junctions composed of connexin 43. Therefore, in a new project we started to investigate the mechanisms by which cardiomyocyte cohesion is regulated and how desmosomal contacts control gap junctions.

Regulation of gap junction function and Connexin 43 expression by cytochrome P450 ..

An open gap junction, composed of six identical connexin proteins

"Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in …

Gap Junction Intercellular Communication (GJIC) has been hypothesized to play a critical role in the coordination of bone development by the ability of gap junctions to permit diffusion of ions, metabolites and small signaling molecules. To measure functional activity of Cx43, GJIC was measured in stable CYPOR knock-down MG63 and 2T3 cells to investigate how CYPOR regulates intercellular communication through Cx43 channels. Transfer of the dye, Alexa-488 (MW 640 Daltons), from the injected cell (donor cell) to the surrounding cells (recipient cells) was monitored in both wild type and CYPOR knock-down MG63 and 2T3 cells by taking images over time (). Wild Type MG63 and 2T3 cells showed a rapid transfer of Alexa-488 from the donor cells to the recipient cells compared to the knock-down cells, suggesting that GJIC is reduced in cells in which CYPOR is knocked-down by shRNA. Unpaired hemichannels can also communicate with the extracellular milieu providing an alternative mechanism for connexin function. Hemichannels have been shown to regulate the release of NAD+, ATP and prostaglandinE2 (PGE2) [,]. To test for hemichannel function, ethidium bromide dye uptake assay performed on wild-type and CYPOR knock-down 2T3 cells (). The CYPOR knock-down cells showed a 30% reduction in hemichannel activity compared to the wild-type 2T3 cells. Taken together, these data show that the decreased expression of Cx43 protein upon CYPOR knock-down affects both the gap junction as well as the hemichannel function.

Hereditary Motor Sensory Neuropathies: Charcot …

100+ Up Regulation Of Connexin43 In Ser364 Of Connexin43 And The Upregulation Of Gap Junction Assembly,Ser364 Of Connexin43 And

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