Cx43 mRNA Levels Research Articles

Differential expression and regulation of connexin-43 and cell-cell coupling in myocytes from the circular and longitudinal layers of bovine myometrium.

The expression and localization of the gap junction protein connexin-43 (Cx-43) as well as functional coupling were studied in myocytes from the two layers of the bovine myometrium: the circular and the longitudinal layers. Intercellular communication (measured by Lucifer yellow dye transfer through gap junctions) was more intense in the circular than in the longitudinal layer of the bovine myometrium. The circular layer also exhibited a greater degree of punctuate immunofluorescence to Cx-43. Myocytes from the circular layer expressed more Cx-43 messenger RNA (mRNA; 2.38 +/- 0.46, Cx-43 over 18S RNA) than the longitudinal layer (1.46 +/- 0.48, Cx-43 over 18S RNA; P < 0.05). The modulation of Cx-43 expression by sex steroids in the two myometrial layers was tested using a pure steroidal antiestrogen, EM-139. In myocytes from the circular layer, the level of Cx-43 mRNA was decreased after treatment with 0.1 microM EM-139 (1.37 +/- 0.25, Cx-43 over 18S RNA) compared to that in untreated cells (2.38 +/- 0.46, Cx-43 over 18S RNA), representing a 40% inhibition. In parallel, cell-cell coupling and the amount of Cx-43 protein were also reduced after antiestrogen treatment. In contrast, treatment of cells from the longitudinal layer with the antiestrogen did not significantly affect the level of Cx-43 mRNA, protein, or cell-cell coupling. These data demonstrate that Cx-43 protein and mRNA are expressed and regulated differentially in myocytes from the circular and longitudinal layers of bovine myometrium. Furthermore, the circular myometrial layer may represent a preferential target for estrogen regulation of the biochemical and mechanical processes controlling contractility.

Differential expression and regulation of connexin-43 and cell-cell coupling in myocytes from the circular and longitudinal layers of bovine myometrium

The expression and localization of the gap junction protein connexin-43 (Cx-43) as well as functional coupling were studied in myocytes from the two layers of the bovine myometrium: the circular and the longitudinal layers. Intercellular communication (measured by Lucifer yellow dye transfer through gap junctions) was more intense in the circular than in the longitudinal layer of the bovine myometrium. The circular layer also exhibited a greater degree of punctuate immunofluorescence to Cx-43. Myocytes from the circular layer expressed more Cx-43 messenger RNA (mRNA; 2.38 +/- 0.46, Cx-43 over 18S RNA) than the longitudinal layer (1.46 +/- 0.48, Cx-43 over 18S RNA; P < 0.05). The modulation of Cx-43 expression by sex steroids in the two myometrial layers was tested using a pure steroidal antiestrogen, EM-139. In myocytes from the circular layer, the level of Cx-43 mRNA was decreased after treatment with 0.1 microM EM-139 (1.37 +/- 0.25, Cx-43 over 18S RNA) compared to that in untreated cells (2.38 +/- 0.46, Cx-43 over 18S RNA), representing a 40% inhibition. In parallel, cell-cell coupling and the amount of Cx-43 protein were also reduced after antiestrogen treatment. In contrast, treatment of cells from the longitudinal layer with the antiestrogen did not significantly affect the level of Cx-43 mRNA, protein, or cell-cell coupling. These data demonstrate that Cx-43 protein and mRNA are expressed and regulated differentially in myocytes from the circular and longitudinal layers of bovine myometrium. Furthermore, the circular myometrial layer may represent a preferential target for estrogen regulation of the biochemical and mechanical processes controlling contractility.

Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos.

Expression of the myometrial gap junction protein connexin-43 (Cx-43) increases dramatically with the onset of labor in association with an increase in the plasma estrogen to progesterone ratio. Moreover, we reported that the levels of messenger RNA (mRNA) and protein encoded by the Cx-43 gene are regulated positively by estrogen and negatively by progesterone in both pregnant and nonpregnant rats. However, although the Cx-43 gene has been reported to be responsive to estrogen, it does not contain a palindromic estrogen response element. It does, however, contain a consensus activator protein-1 (AP-1)-binding site that may be of significance, as estrogen has been reported to increase expression of the AP-1 proteins, Fos and Jun, in the uterus. Therefore, we tested the hypothesis that the increase in Cx-43 mRNA levels in the myometrium during labor and after estrogen administration is mediated indirectly through induction of trans-activating factors, of which Fos and Jun are putative candidates. Treatment of nonpregnant ovariectomized rats with cyclohexamide blocked the estrogen-induced increase in Cx-43 mRNA in the myometrium, suggesting that this action of estrogen requires newly synthesized protein. Estrogen treatment alone induced an increase in mRNA encoding c-fos and c-jun in nonpregnant rats, which preceded by 2-3 h an increase in Cx-43 mRNA. Labor was also associated with a coincident expression of c-fos and Cx-43 in the myometrium, although there was no change in c-jun mRNA levels during this period. These associative data were strengthened by our observation that in paradigms in which labor was induced prematurely by ovariectomy or blocked by treatment with progesterone, changes in c-fos expression were closely matched by changes in Cx-43 mRNA. These data support our hypothesis that estrogen (and, by extension, labor-)-induced increases in Cx-43 mRNA are mediated indirectly through newly synthesized trans-activating factors. Moreover, the temporal correlation between the expression of c-fos and Cx-43 and the presence of AP-1 cis-acting elements within the Cx-43 promoter suggest that c-fos may be one of these trans-activating proteins.

Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos

Expression of the myometrial gap junction protein connexin-43 (Cx-43) increases dramatically with the onset of labor in association with an increase in the plasma estrogen to progesterone ratio. Moreover, we reported that the levels of messenger RNA (mRNA) and protein encoded by the Cx-43 gene are regulated positively by estrogen and negatively by progesterone in both pregnant and nonpregnant rats. However, although the Cx-43 gene has been reported to be responsive to estrogen, it does not contain a palindromic estrogen response element. It does, however, contain a consensus activator protein-1 (AP-1)-binding site that may be of significance, as estrogen has been reported to increase expression of the AP-1 proteins, Fos and Jun, in the uterus. Therefore, we tested the hypothesis that the increase in Cx-43 mRNA levels in the myometrium during labor and after estrogen administration is mediated indirectly through induction of trans-activating factors, of which Fos and Jun are putative candidates. Treatment of nonpregnant ovariectomized rats with cyclohexamide blocked the estrogen-induced increase in Cx-43 mRNA in the myometrium, suggesting that this action of estrogen requires newly synthesized protein. Estrogen treatment alone induced an increase in mRNA encoding c-fos and c-jun in nonpregnant rats, which preceded by 2-3 h an increase in Cx-43 mRNA. Labor was also associated with a coincident expression of c-fos and Cx-43 in the myometrium, although there was no change in c-jun mRNA levels during this period. These associative data were strengthened by our observation that in paradigms in which labor was induced prematurely by ovariectomy or blocked by treatment with progesterone, changes in c-fos expression were closely matched by changes in Cx-43 mRNA. These data support our hypothesis that estrogen (and, by extension, labor-)-induced increases in Cx-43 mRNA are mediated indirectly through newly synthesized trans-activating factors. Moreover, the temporal correlation between the expression of c-fos and Cx-43 and the presence of AP-1 cis-acting elements within the Cx-43 promoter suggest that c-fos may be one of these trans-activating proteins.

Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells.

The mechanism by which all-trans retinoic acid (RA) stimulates gap junctional intercellular communication (GJIC) in the rat liver epithelial cell line. IAR203, was investigated. When RA, at 0.1 microM for 24-48 h, enhanced the dye transfer in IAR203 cells (x 1.4), it increased the amount of connexin43 (Cx43) in the cell-cell contact regions of the plasma membrane, as evidenced by analysis by Western blot and by immunofluorescence. It had no effect on the level of Cx43 mRNA. Freeze-fracture analysis of the size of gap junctions revealed an increase of the proportion of small gap junctions in RA-treated cells. We conclude that, in IAR203 cells, RA stimulates GJIC by acting at the post-transcriptional level of Cx43 regulation. The possibility that RA acts indirectly on the regulation of Cx43 expression, and increases the half-life of Cx43 by inducing adhesion molecules is discussed.

The expression of gap junctional proteins during different stages of mouse skin carcinogenesis.

The loss or alteration of gap junctional intercellular communication (GJIC) has long been proposed to play an important role in the process of carcinogenesis. In this study we examined the expression of three gap junction proteins, connexins (Cx)26, 43 and 31.1, in mouse hyperplastic skin, papillomas and squamous cell carcinomas (SCC). Tumors were induced in SENCAR mice by either of two initiation/promotion protocols: 7,12-dimethylbenz-[a] anthracene (DMBA)/12-O-tetradecanoyl-phorbol-13-acetate (TPA) or DMBA/benzoyl-peroxide (BzPo). Keratinocytes in adult mouse skin expressed Cx31.1 and Cx43 but did not express Cx26. Skin hyperplasia induced by one topical application of TPA was accompanied by hyperexpression of both Cx26 and Cx43. In addition, TPA significantly inhibited the expression of Cx31.1. After repetitive application. Connexin 26 and Cx43 were hyperexpressed in most of the papillomas studied (20-40 weeks after initiation). However, in some late papillomas, immunostaining revealed a focal loss of Cx26. Immunostaining of mouse skin SCC revealed decreased Cx43 and Cx26 levels in 65% and 85% of cases respectively. The high levels of Cx26 and Cx43 mRNA in most of the SCC did not correlate with the decreased abundance or disappearance of Cx26 and Cx43 immunoreactive spots from tumor plasma membranes. Thus, the expression of these two connexins in SCC was impaired at the post-translation level. Cx31.1 expression was strongly inhibited during all stages of carcinogenesis. Taken together, our results suggest that three different connexin genes are differentially regulated during mouse skin carcinogenesis and the decrease of connexin expression may be an important marker of skin tumor expression.

Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone.

One important role of the junctional communication in the ovarian follicle is to mediate transmission of cAMP, the regulatory signal that maintains the oocyte in meiotic arrest. Luteinizing hormone (LH) interrupts cell-to-cell communication within the ovarian follicle, leading to a decrease in intraoocyte concentrations of cAMP followed by resumption of meiosis. Our experiments were directed at exploration of mechanisms involved in the LH-induced communication breakdown in the preovulatory ovarian follicle. Immunofluorescence and Western blot analysis, using highly specific antibodies, showed that connexin-43 (Cx43), the ovarian gap junction protein, is present in the cytoplasmic membranes of the follicular cells in multiple phosphorylated forms. The relative amounts of the different forms of Cx43 vary in response to LH: short time exposure (10 min) stimulated phosphorylation of Cx43 followed by immediate dephosphorylation, while longer incubations (8 and 24 h) with this hormone resulted in elimination of the protein. Forskolin mimicked the LH-induced phosphorylation/dephosphorylation, as well as the decrease of Cx43 protein level. A gonadotropin-releasing hormone analog (GnRHa) also induced an immediate phosphorylation/dephosphorylation of Cx43 and a later reduction of the amount of Cx43. The direct PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced phosphorylation of Cx43 that was completely blocked by the protein kinase C inhibitor, staurosporine. This kinase inhibitor partially interfered with LH, but not forskolin-induced phosphorylation of Cx43. Analysis of the effect of LH on Cx43 gene expression revealed a significant decrease (45%) in Cx43 mRNA level at 24 h of incubation. A drop of Cx43 mRNA was also induced by GnRHa. Our results suggest that the LH-induced gating mechanism of the gap junctions in rat ovarian follicles is comprised of two steps: the immediate response is represented by a change in the phosphorylation state of the Cx43 protein, and the later response is manifested by a reduction of Cx43 protein level, due to attenuation of its gene expression. Phosphorylation of Cx43 may occur through PKA-, as well as PKC-dependent pathways.

Modulation of Gap Junctions in Senescent Endothelial Cells

Gap junction-mediated intercellular communication (GJIC) was decreased in senescent human umbilical vein endothelial cells (HUVEC), as detected by Gap-Frap studies. The molecular basis of this reduction and the effects of the calcium ionophore A23187 and epidermal growth factor (EGF) on young and old HUVEC have been investigated. Northern and Western analyses reveal that the levees of both cx43 (connexin 43) messenger RNA and protein decline as HUVEC age in vitro. While both young and senescent cells responded immediately to increases in intracellular calcium concentrations, only young cells produced a dose-dependent decrease in cell coupling in response to the addition of exogenous EGF. The down-regulation of cx43 mRNA and protein levels in senescent endothelial cells suggests that GJIC might play a role in the aging process. The inability of senescent cells to down-regulate gap junctions in response to EGF reflects a defect in the regulatory mechanism of gap junction activity in senescent cells.

Expression of connexin 43 gap junction messenger ribonucleic acid and protein during follicular atresia.

The process of atresia is an all-or-none phenomenon in that an entire follicle either undergoes atresia or continues along the developmental pathway. The absence of pockets of atresia adjacent to healthy areas of granulosa cells suggests the existence of a coordinating influence within the entire follicular unit during the process of atresia. Gap junctions interconnect the granulosa cells and the oocyte in the ovarian follicle, forming a metabolic syncytium. Intercellular communication provided via the gap junctions may play a role in coordinating the process of atresia. This study addresses the potential hormonal regulation of the gap junction gene during the process of atresia. Immature female rats were given an estradiol (E2) implant to induce follicular development, and after 48 h the E2 was withdrawn to induce atresia. The ovary is an extremely heterogeneous tissue with multiple cell types and many follicles at different stages of development. Therefore, in situ hybridization and immunocytochemistry were ideal techniques to localize gap junction gene expression precisely to specific cells in the ovary. Observations resulting from these studies revealed that while the granulosa cells of healthy, developing, pre-antral and antral follicles expressed large amounts of connexin 43 (cx 43) gap junction mRNA and protein, this expression was greatly reduced in atretic follicles. In the follicles undergoing atresia, the levels of cx 43 gap junction mRNA and protein were reduced as early as 6 h after the withdrawal of E2. The levels of cx43 mRNA and protein continued to decrease as atresia progressed, and at 11 h after withdrawal of E2 very little cx43 mRNA or protein was seen. These results indicate that gap junction mRNA and protein are decreased in association with atresia and support the hypothesis that a loss of gap junctional communication plays a coordinating role in the process of atresia.

Effect of DDT on hepatic gap junctional intercellular communication in rats.

The effects of in vivo exposure to DDT on hepatic gap junctional intercellular communication (GJIC) and connexin gene/protein expression in Sprague-Dawley rats were examined by in vivo/in vitro dye-transfer assay, immunohistochemical staining, and by Western and Northern blot analyses. In the dose-response study, three dose levels of DDT (5, 25 and 50 mg/kg/day) were administered orally to rats once a day for 2 weeks. The average size of the dye spread after injection of Lucifer Yellow and the area of Cx32 spots per hepatocyte decreased in a dose-dependent manner, but there was no effect on the number of Cx32 spots per hepatocyte. In the time-course study, DDT (50 mg/kg/day) was administered orally once a day for up to 6 weeks. Hepatic GJIC decreased at week 1 but recovered at week 6. The average area of Cx32 spots per hepatocyte gradually decreased at weeks 2 and 4, and remained at the same level at week 6, correlating with the decreased Cx32 protein level in plasma membranes. The average area of Cx26 spots per hepatocyte in the peripheral zones clearly decreased at week 1, but quickly recovered at week 2 and increased at week 6; however, no clear change of the Cx26 protein level in plasma membranes was observed. No changes of Cx32 and Cx26 mRNA levels were observed in DDT groups. These results suggest that DDT, a liver tumor-promoting agent, inhibits hepatic GJIC in vivo dose-dependently in rats and that aberrant Cx32 and Cx26 protein expression and/or localization may be responsible for this effect.

Expression of mRNA for the gap‐junctional protein connexin43 in human colonic tissue is variable in response to β‐carotene supplementation

Increased expression of the gap-junctional protein connexin43 (Cx43) is reported to be increased in mouse and human dermal fibroblasts in vitro in response to beta-carotene treatment. In the present study, we determined the level of Cx43 mRNA expression in colonic mucosa from normal subjects and subjects with a prior history of colonic polyps or cancer before and after three months of administration of a placebo or beta-carotene. RNA was reverse transcribed and used in a polymerase chain reaction assay employing primers selected from the human Cx43 gene sequence. Cx43 mRNA expression was normalized on the basis of beta 2-microglobulin mRNA expression. The beta-carotene concentration in colonic mucosa, as well as serum and diet, was also determined to establish a correlation between Cx43 expression and beta-carotene concentration. In an initial analysis of samples collected from 10 subjects before supplementation, the quantity of Cx43 mRNA was variable and did not correlate with beta-carotene intake or the concentration of beta-carotene in tissue or serum. In samples collected at zero and three months from eight subjects who were controls or received a placebo, there was no correlation between Cx43 mRNA level and tissue or serum beta-carotene concentration. In samples collected from subjects before and after three months of beta-carotene supplementation, there was a significant increase in tissue and serum beta-carotene concentration in all subjects and an increase in Cx43 mRNA expression after supplementation relative to baseline in four of six samples. The high variability in Cx43 expression indicates that induction of Cx43 mRNA expression is not solely dependent on the concentration of beta-carotene in diet, serum, or tissue. However, the results from subjects supplemented with beta-carotene suggest that induced expression may occur in colonic tissue of some individuals. some individuals.

Multiple mechanisms are responsible for altered expression of gap junction genes during oncogenesis in rat liver.

Although several abnormalities in gap junction (GJ) structure and/or function have been described in neoplasms, the molecular mechanisms responsible for many of the alterations remain unknown. The identification of a family of GJ proteins, termed connexins, prompted this study of connexin32 (Cx32), connexin26 (Cx26) and connexin43 (Cx43) expression during rat hepatocarcinogenesis. Using antibody, cDNA and cRNA probes, we investigated connexin mRNA and protein expression in preneoplastic and neoplastic rat livers. In normal liver, Cx32 is expressed in hepatocytes throughout the hepatic acinus, Cx26 is restricted to periportal hepatocytes, and Cx43 is expressed by mesothelial cells forming Glisson's capsule. Most preneoplastic altered hepatic foci generated by diethylnitrosamine (DEN) initiation and either phenobarbital (PB) or 2,3,7,8-dichlorodibenzo-p-dioxin (TCDD) promotion exhibited decreased Cx32 or increased Cx26 staining. Foci from either protocol failed to display Cx43 immunoreactivity. In the majority of PB-promoted foci, Cx32 immunoreactivity decreased independently of changes in mRNA abundance. Continuous thymidine labeling, following cessation of PB promotion, showed that downregulation of Cx32 staining is reversible in foci that are promoter-dependent for growth, but irreversible in lesions that are promoter-independent for growth. Hepatic neoplasms from rats initiated with DEN and promoted with PB or TCDD also displayed modified connexin expression. While all 24 neoplasms studied were deficient in normal punctate Cx32 and Cx26 staining, altered cellular localization of these proteins was apparent in some tumors. Immunoblotting of crude tissue extracts revealed that neoplasms with disordered Cx32 staining showed immunoreactive bands with altered electrophoretic mobility. These observations show that hepatomas may downregulate Cx32 expression through changes in the primary structure of Cx32 or by post-translational modifications. Northern blotting of total tumor mRNAs failed to demonstrate consistent changes in the abundance of Cx32, Cx26 or Cx43 transcripts. Some tumors expressed steady-state transcripts without observable immunoreactivity, indicating that some hepatomas downregulate connexin immunoreactivity independently of mRNA abundance. Increased levels of Cx43 mRNA and protein were found in several neoplasms, but immunostaining was always localized to nonparenchymal cells. Areas of bile duct proliferation and cholangiomas displayed Cx43 staining, whereas, cholangiocarcinomas were deficient in immunoreactivity. These findings show that alterations in the expression of connexins, by either downregulation or differential induction, represent common modifications during hepatocarcinogenesis. Although our results imply that connexins represent useful markers for the boundary between tumor promotion and progression, preneoplastic and neoplastic rat hepatocytes fail to use a common mechanism to modify connexin expression.

Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone.

In the rat, transcripts encoding the myometrial gap junction protein, connexin-43 (Cx-43), increase dramatically with the onset of labor in association with an increase in the ratio of estrogen to progesterone in plasma. We examined whether the level of Cx-43 transcripts might be regulated by these steroids in the rat myometrium. Administration of progesterone to late pregnant rats abolished the more than 12-fold increase in transcripts seen in control rats at term and blocked delivery of the fetuses. Treatment of rats on day 15 of gestation (when Cx-43 mRNA levels are low) with the progesterone antagonist RU486 resulted in a significant (2.5-fold) increase in transcripts within 9 h, with the maximal (5.6-fold) increase occurring between 24-48 h, and preterm delivery occurring between 48-72 h. Administration of a single dose of 17 beta-estradiol (5 micrograms, sc) to nonpregnant rats resulted in a significant increase in Cx-43 transcripts within 3 h; these levels were increased 3-fold between 6-24 h, before falling to lower levels by 48 h. Progesterone (4 mg, sc) administration at the same time as estradiol significantly attenuated the estradiol response. Chronic estradiol administration (5 micrograms/12 h for 36 h) failed to maintain elevated levels of Cx-43 transcripts beyond 36 h. Administration of progesterone 12 h after estradiol prematurely reduced the level of Cx-43 transcripts. These data demonstrate that steroid hormones can modulate the level of steady state transcripts of Cx-43 during pregnancy in association with changes in uterine contractile activity. Furthermore, the data from the nonpregnant studies suggest that the levels of transcripts are regulated positively by estradiol and negatively by progesterone.

Transcription of the gene for the gap junctional protein connexin43 and expression of functional cell-to-cell channels are regulated by cAMP.

We investigated the mechanism by which cyclic AMP (cAMP) induces gap junctional communication via cell-to-cell channels in a communication-deficient rat Morris hepatoma cell line. We found that under basal conditions, the cells transcribe cx43 at a low level but do not transcribe cx26 or cx32. Elevation of intracellular cAMP, which induced communication, increased cx43 mRNA 15- to 40-fold and the rate of cx43 transcription 6-fold. Cx43 protein was detected by immunostaining in junctions of only those cells in which communication had been induced. We found the regulation by cAMP also in other cell lines; namely, in those with a low basal level of cx43 mRNA.