Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA.

Fuhlbrigge, Robert C.; Fine, Steven M.; Unanue, Emil R.; Chaplin, David

doi:10.1073/pnas.85.15.5649

Cited by 152 publications

(71 citation statements)

References 22 publications

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“…Both p6xOSE2-and p6xOSE2mut-luciferase constructs were provided by Dr. Gerard Karsenty (Columbia University, New York, NY). The pSFFV-Cx43 construct (Beyer et al, 1987), which contains a full-length cDNA for rat Cx43 in the pSFFV-neo backbone plasmid (described in Fuhlbrigge et al, 1988), was provided by Dr. Thomas Steinberg (Washington University, St. Louis, MO). This construct has previously been shown to increase electrical (Veenstra et al, 1992) and chemical (Steinberg et al, 1994) coupling among cells.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

Lima

Niger

Hebert

et al. 2009

MBoC

100

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In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKC␦) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKC␦ translocates to the nucleus, PKC␦ and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKC␦ and Runx2 function. INTRODUCTIONBone formation and remodeling is a tightly organized and dynamic process that requires the coordinated action of osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. It is hypothesized that osteoblasts and osteocytes coordinate their activities, at least in part, through direct cell-to-cell communication through gap junctions. Gap junctions are composed of connexins, a family of transmembrane proteins that constitute the intercellular gap junction channels (Beyer et al., 1990). Six connexins assemble to make up the gap junction hemichannel on the plasma membrane of one cell, which docks with a hemichannel on an adjacent cell to form an aqueous gap junction channel. The resultant gap junctions provide direct conduits for the passage of ions and other low molecular weight molecules, including second messengers, among cells.The gap junction protein connexin43 (Cx43) is abundantly expressed in both osteoblasts and osteocytes, where it has been hypothesized to transmit hormonal signals, mechanical load, and growth factor cues among cells in order to coordinate the synthesis of new bone (reviewed in Stains and Civitelli, 2005a;Jiang et al., 2007). Genetic ablation of gja1, the gene encoding Cx43, in mice leads to a severe delay in the ossification of both intramembranous and endochondral derived skeletal elements during embryonic development (Lecanda et al., 2000). The bones of these animals are remarkably brittle, and the osteoblasts isolated from the Cx43 null animals are dysfunctional, with reduced osteogenic and mineralizing capacity (Lecanda et al., 2000). These Cx43-deficient mice die at birth because of a defect in cardiac f...

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Section: Plasmid Constructsmentioning

confidence: 99%

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

Lima

Niger

Hebert

et al. 2009

MBoC

100

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“…The mammalian expression vector, pSFFV-neo, and the Bcl-2 expression construct, pSFFV-hBcl-2, have been described before (Fuhlbrigge et al, 1988;Hockenbery et al, 1990). The antisense bcl-2 expression plasmid was constructed using a different expression vector, pZeoSV vector (Invitrogen, CAT#V850-01), with the Bcl-2 cDNA inserted at the EcoRI site in the 3 0 to 5 0 direction (Mao et al, 2001).…”

Section: Establishment Of Stable Expression Cell Linesmentioning

confidence: 99%

Human Bcl-2 activates ERK signaling pathway to regulate activating protein-1, lens epithelium-derived growth factor and downstream genes

et al. 2004

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The proto-oncogene, bcl-2, has various functions besides its role in protecting cells from apoptosis. One of the functions is to regulate expression of other genes. Previous studies have demonstrated that Bcl-2 regulates activities of several important transcription factors including NFjB and p53, and also their downstream genes. In our recent studies, we reported that Bcl-2 substantially downregulates expression of the endogenous aB-crystallin gene through modulating the transcriptional activity of lens epithelium-derived growth factor (LEDGF). In the present communication, we report that human Bcl-2 can positively regulate expression of the proto-oncogenes c-jun and c-fos. Moreover, it enhances the DNA binding activity and transactivity of the activating protein-1 (AP-1). Furthermore, we present evidence to show that Bcl-2 can also activate both ERK1 and ERK2 MAP kinases. Inhibition of the activities of these kinases or the upstream activating kinases by pharmacological inhibitors or dominant-negative mutants abolishes the Bcl-2-mediated regulation of AP-1, LEDGF and their downstream genes. Together, our results demonstrate that through activation of the ERK kinase signaling pathway, Bcl-2 regulates the transcriptional activities of multiple transcription factors, and hence modulates the expression of their downstream genes. Thus, our results provide a mechanism to explain how Bcl-2 may regulate expression of other genes.

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“…Wild-type human Cx50 (Shiels et al, 1998;Pal et al, 1999) was subcloned into the EcoRI site of the eukaryotic expression vector pSFFV-neo (Fuhlbrigge et al, 1988). Carboxyl terminal deletion mutants of Cx50 were obtained by PCR using a sense primer, 5′-ggaattcaccatgggcgactgg-3′, and two antisense primers, 5′-ccggaattctcactcggtcaagggg-3′ and 5′-ccggaattctcaaggcagtgggctgg-3′ to generate Cx50 polypeptides that ended at valine-284 (Cx50V284stop) and alanine-294 (Cx50A294stop), respectively (Fig.…”

Section: Preparation Of Wild-type Cx50 and C-terminus-truncated Cx50 mentioning

confidence: 99%

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

Berthoud

Beyer

et al. 2002

Journal of Membrane Biology

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Gap junction channels formed by connexin 50 (Cx50) are critical for maintenance of lens transparency. Because the C-terminus of Cx50 can be cleaved post-translationally, we hypothesized that channels formed by the truncated Cx50 exhibit altered properties or regulation. We used the dual whole-cell patch-clamp technique to investigate the macroscopic and single-channel properties of gap junctional channels formed by wild-type human Cx50 and a truncation mutant (Cx50A294stop) after transfection of N2A cells. Our results show that wild-type Cx50 formed functional gap junctional channels. The macroscopic G jss -V j relationship was well described by a Boltzmann equation with A of 0.10, V 0 of 43.8 mV and G jmin of 0.23. The single-channel conductance was 212 ± 5 pS. Multiple long-lasting substates were observed with conductances ranging between 31 and 80 pS. Wild-type Cx50 gap junctional channels were reversibly blocked when pH i was reduced to 6.3. Truncating the C-terminus at amino acid 294 caused a loss of pH i sensitivity, but there were no significant changes in single-channel current amplitude or G jss -V j relationship. These results suggest that the C-terminus of human Cx50 is involved in pH i sensitivity, but has little influence over singlechannel conductance, voltage dependence, or gating kinetics.

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Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA.

Cited by 152 publications

References 22 publications

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

Human Bcl-2 activates ERK signaling pathway to regulate activating protein-1, lens epithelium-derived growth factor and downstream genes

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

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