Role of PKC and MAP kinase in EGF- and TPA-induced connexin43 phosphorylation and inhibition of gap junction intercellular communication in rat liver epithelial cells

Rivedal, Edgar; Opsahl, Hanne

doi:10.1093/carcin/22.9.1543

Cited by 122 publications

(100 citation statements)

References 55 publications

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“…In our previous study, ERK-and p38 MAP kinase inhibitors were found to prevent the inhibition of GJIC induced by TPA and H 2 O 2 [1,9]. TPA has been reported to induce the inhibition of GJIC [11,18], and the inhibition of GJIC by TPA which likely contributes to alterations of growth, differentiation, and apoptosis [21]. Furthermore, differential regulation of Cx43 expres- Fig.…”

Section: Discussionmentioning

confidence: 92%

Role of Gap Junctional Intercellular Communication (GJIC) through p38 and ERK1/2 Pathway in the Differentiation of Rat Neuronal Stem Cells

Yang

Cho

Ahn

et al. 2005

The Journal of Veterinary Medical Science

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ABSTRACT. Gap junctional intercellular communications (GJIC) contributes to neural function in development and differentiation of CNS. In this study, we have investigated the expression of GJIC during the differentiation of neuronal stem cells and 12-Ø-tetradecanoylphorbol-13-acetate (TPA)-induced neuronal stem cell-derived cells from rat brain. During neuronal stem cell differentiation, expressions of Cx43 and 32 were increased for the duration of 72 hr, however the effect were decreased on the 7d. In the neuronal stem cell-derived cells, pretreatments with p38 MAP kinase inhibitor, SB203580, and MEK inhibitor, PD98059, could protect GJIC against TPA-induced inhibition of GJIC. Our data suggest that GJIC plays an important role during neuronal stem cell differentiation, and ERK1/2 and p38 MAP kinase signaling pathway may be closely related functionally to regulate gap junction in rat neuronal stem cell-derived cells. Neuronal stem cells are able to differentiate into astrocytes, neurons, and oligodendrocytes [3,10,23], when given the appropriate signals [2,7]. During the stage where the brain develops, gap junctional intercellular communication (GJIC) allows the diffusional exchange of ions and metabolites for communication between neighboring cells [8,14,20]. Connexin 32 and 43 were expressed in the neurons of adult rat or mouse [15,17], and Cx43 was the most abundant gap junction protein in the CNS [4,5]. However, no study has yet been performed to the role of gap junctions in rat neuronal stem cell differentiation. In the present study, we have examined protein expression of Cx43 and Cx32 during the neuronal stem cell differentiation and induction of Cx43 and Cx32. This can be elicited in neuronal stem cell-derived cells by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and this induction showed that it can be prevented by the p38 MAP-Kinase inhibitor SB203580 and the MEK inhibitor PD98059. MATERIALS AND METHODSCell culture: Neuronal stem cells were prepared by culturing cells from cerebrum of embryonic day 17 (E17) Sprague-Dawley rat (Bio Genomics, Korea) fetus. Briefly, fresh cerebrums were dissociated in the presence of trypsin and DNase I and planted on a Petri dish (Nunc, IL). Cells were seeded at a density of 1~5 × 10 6 cells/ml in a mixture (1:1) of Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12; Gibco) replenished with 2% B27 (Gibco), gentamycin (50 µg/ml), anti-PPLO (pleuro-pneumonia-like organism) reagent (100 µg/ml), recombinant human basic fibroblast growth factor (bFGF 20 ng/ml, Sigma), and epidermal growth factor (10 ng/ml).Scrape loading/ dye transfer (SL/DT) assay: Cells were treated with TPA for 1 hr. The GJIC assay was conducted at non-cytotoxic dose levels of the samples, as determined by the MTT assay. Following incubation, the cells were washed twice with 2 ml of PBS, Lucifer yellow was added to the washed cells and three scrapes were made with a surgical steel-bladed scalpel at low light intensities. These three scrapes were performed to ensure that the scrape travers...

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Section: Discussionmentioning

confidence: 92%

Role of Gap Junctional Intercellular Communication (GJIC) through p38 and ERK1/2 Pathway in the Differentiation of Rat Neuronal Stem Cells

Yang

Cho

Ahn

et al. 2005

The Journal of Veterinary Medical Science

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“…, not on tyrosine, but serine amino acids (Hossain, Ao, & Boynton, 1998a;Kanemitsu & Lau, 1993;Lau, Kanemitsu, Kurata, Danesh, & Boynton, 1992;Rivedal & Opsahl, 2001). The disruption of gap junctional communication appeared to occur independently of observable changes in gap junction plaques (Lau et al, 1992).…”

Section: Receptor Protein Tyrosine Kinases-in Many Cases Activation mentioning

confidence: 98%

The effects of connexin phosphorylation on gap junctional communication

Lampe

Lau

2004

The International Journal of Biochemistry & Cell Biology

543

463

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Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the "connexin" gene family. In most cases examined, connexins are modified posttranslationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34 cdc2 /cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60 src kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication.

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“…Several studies have focused on using growth factors and PMA in combination with MAPK and PKC inhibitors to correlate changes in Cx43 isoform migration with shutdown of gap junctional communication. In one study, IAR6.1 cells, which endogenously express Cx43 and make P2 in resting cells, exhibited a decrease in gap junctional communication and a migration shift in response to both PMA and EGF (Rivedal & Opsahl, 2001). In these cells, inhibition of ERK1/2 but not PKC inhibition could inhibit the migration shift in response to PMA and EGF, although it did not reverse PMA induced inhibition of gap junctional communication.…”

Section: Induced Phosphorylation Can Lead To a Distinct P2mentioning

confidence: 99%

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

2007

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Connexin43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane ultimately forming into a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in "phosphorylation" based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here we indicate how phosphospecific and epitope specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, 328, and/or 330 is necessary to form a P2 isoform and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO cells but not MDCK cells. The shift was dependent on MAPK activity but not phosphorylation at S279/282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to be able to sort out the critical signaling pathways that regulate gap junction function.

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Role of PKC and MAP kinase in EGF- and TPA-induced connexin43 phosphorylation and inhibition of gap junction intercellular communication in rat liver epithelial cells

Cited by 122 publications

References 55 publications

Role of Gap Junctional Intercellular Communication (GJIC) through p38 and ERK1/2 Pathway in the Differentiation of Rat Neuronal Stem Cells

Role of Gap Junctional Intercellular Communication (GJIC) through p38 and ERK1/2 Pathway in the Differentiation of Rat Neuronal Stem Cells

The effects of connexin phosphorylation on gap junctional communication

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

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