Differential control of connexin-32 and connexin-43 expression in thyroid epithelial cells: evidence for a direct relationship between connexin-32 expression and histiotypic morphogenesis.

Thyroid epithelial cells cultured either as a monolayer or in the form of follicles, rapidly reconstitute functional gap junctions (Gj). We previously reported that the thyroid Gj gating is regulated by TSH. We have now performed molecular analyses of Gj proteins 1) to detect the connexin(s) (Cx) that is expressed in thyroid epithelial cells, 2) to determine whether the expression of Cx is hormonally regulated, and 3) to analyze the relationship between Cx expression and histiotypic morphogenesis, i.e. folliculogenesis. Studies were carried out on thyrocytes freshly isolated from the gland and on corresponding thyrocytes after 1-7 days in culture as monolayers or in the form of reconstituted follicles. The Cx gene transcription products were analyzed by Northern blot using specific complementary DNA probes for Cx26, Cx32, and Cx43. Cx proteins were identified and estimated by Western blot and indirect immunofluorescence using polyclonal antipeptide antibodies. Cx32 and Cx43 proteins and their corresponding messenger RNA (mRNA) were detected in thyrocytes freshly isolated from the gland. Thyrocytes contained a high amount of the 1.6-kilobase Cx32 mRNA and only traces of the 3-kilobase Cx43 transcript. No Cx26 transcripts could be detected. Thyrocytes cultured at a density of 0.2-0.5 x 10(6) cells/cm2 in the absence of TSH formed monolayers. Surprisingly, monolayer cells lost Cx32 protein within 24 h, and their Cx32 mRNA content decreased from high to barely detectable levels; Cx32 protein was no longer detected throughout the 1-week culture period. On the contrary, Cx43 mRNA and Cx43 protein rapidly increased in monolayer cells to reach very high levels within 2-4 days. Thyrocytes cultured at the same density, but in the presence of TSH also rapidly lost Cx32, but as soon as they reorganized into follicular structures, reexpressed Cx32 at a level (in terms of protein and mRNA) comparable to that found in cells freshly extracted from the gland. As observed for cell monolayers, reconstituted follicles overexpressed Cx43. The Cx43 protein and Cx43 mRNA contents of cultured thyrocytes were 20- to 50-fold higher than those found in isolated thyrocytes at the outset of culture. When thyrocytes were cultured with TSH, but at a low density (< 0.2 x 10(6) cells/cm2) to prevent follicle formation, a TSH-dependent increase in Cx43 was observed in monolayer cells. However, TSH did not cause any reexpression of Cx32.(ABSTRACT TRUNCATED AT 400 WORDS)

Cooperation of Ito cells and hepatocytes in the deposition of an extracellular matrix in vitro.

Cellular and molecular mechanisms involved in the deposition of extracellular matrix components in both normal and fibrotic liver are still poorly understood. We have investigated the influence of cooperation between Ito cells and hepatocytes in matrix deposition in vitro. Immunoprecipitation of radiolabeled proteins from media of 5-day-old Ito cell primary cultures showed that these cells secreted high levels of the major basement membrane components, ie, collagen IV, laminin, and entactin/nidogen. By immunocytochemistry, precursors of basement membrane components were found intracellularly, but only scarce deposits were seen around the cells. When hepatocytes were added to 2-day-old Ito cell primary cultures, they established close contacts with Ito cells in less than 24 hours and expressed ZO-1, a tight junction-associated protein not detectable in standard hepatocyte culture. Cytochemistry analysis revealed an abundant extracellular matrix deposited over hepatocyte cords and between hepatocytes and Ito cells. Immunocytochemistry studies showed that this matrix contained laminin, fibronectin, and collagens proIII and IV. These data indicate that a high level of matrix protein synthesis by liver cells in vitro is not sufficient to induce extracellular matrix deposition, and that cell-cell interactions are strongly involved in this process. Hepatocyte/Ito cell co-culture, which may reflect the actual situation in vivo, represents a useful tool for studying liver fibrogenesis.

Distribution pattern of connexin 43, a gap junctional protein, during the differentiation of mouse heart myocytes.

In the cardiac muscle, the electrical coupling of myocytes by means of gap (or communicating) junctions, allows the action potentials to be propagated. Connexin 43 (CX 43) is the major constitutive protein of the gap junctions in the mammalian myocardium. In this organ, the abundance of CX 43 and of its messenger, as well as the spatial expression of this protein, are developmentally regulated. These findings are complemented by the results presented in this article, which deals with the distribution of CX 43 in the ventricular myocytes of mouse heart during differentiation, between the 11 days post coitum embryo stage and adulthood. By immunoelectron microscopy experiments on ultrathin sections of cardiac ventricular tissue of one-week-old mouse, we have provided confirmation that the anti-CX 43 antibodies used here specifically recognized the gap junctions. Double labeling immunofluorescence experiments have been undertaken to localize, within the same cells, either CX 43 and desmin, or CX 43 and Con A or WGA receptor sites. From the earliest stage investigated (11 days post coitum) onwards, expression of CX 43 is always associated with desmin-positive cells, that is, with the myocytes. Up to birth, there is in the ventricular wall a gradient of expression of CX 43 which is superimposable on a gradient of expression of desmin. Immunoreactivity to anti-CX 43 and anti-desmin antibodies is high in the sub-endocardial trabeculae and low (or even undetectable for CX 43, in the early stages) in the sub-epicardial cell layers. In the embryonic stages, the expression sites of CX 43 are visible in the form of small dots, whose abundance increases as development proceeds. During these stages, the immunoreactive sites are distributed in a relatively homogeneous pattern throughout the membrane of the myocytes. One week after birth, the CX 43 expression is restricted to the two ends of the myocytes (where the intercalated discs develop), and the adjacent lateral regions. This polarization of CX 43 is more pronounced at the two and three weeks post natal stages and in the fully differentiated ventricular myocytes (adult stage) CX 43 is only present in the intercalated discs.

Immunolocalization of an extracellular domain of connexin43 in rat heart gap junctions.

Antipeptide antibodies directed to residues 55 to 66 (NTQQPGCENVCY) of connexin43 (cx43) specifically recognize this protein on Western blots of intact and urea-split gap junctions isolated from rat heart. These antibodies detect a single protein of 43 kDa, corresponding to cx43, on Western blots of whole fractions of various vertebrate hearts. Immunogold labeling by electron microscopy shows that the epitopes recognized by these antibodies are not localized on the cytoplasmic surfaces of intact gap junctions but only at the edges of these junctions. In urea-split gap junctions the gold particles are seen in the junctional space, associated with the extracellular faces of junctional membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of rat heart gap junctions treated with trypsin show that they are constituted with either two polypeptides of Mr 12,000 and 14,000 or a single polypeptide of Mr 22,000 according to whether the analyses are performed under reducing or non-reducing conditions, respectively. The antibodies directed to residues 55 to 66 of cx43 cross-react with both the 12 and 22 kDa polypeptides. These results suggest that the two protected domains of 12 and 14 kDa which contain the first extracellular loop and a putative second extracellular loop, respectively, are linked by disulfide bonds. In adult rat heart sections analyzed by indirect immunofluorescence the intercalated discs are labeled with antibodies directed to a cytoplasmic carboxy-terminal domain of cx43 (El Aoumari et al., J. Membr. Biol. 115, 229-240 (1990)). The same intercalated discs are also labeled in adjacent sections incubated with the antibodies directed to residues 55 to 66. Two hypotheses might explain these results: either the antibodies have access to the extracellular domain of cx43 molecules localized at the edges of the gap junctions, or cx43 molecules are present in the non-junctional membranes of the intercalated discs.

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.

Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart.

The developmental appearance and spatial distribution pattern of gap junctions were studied in prenatal and adult rat hearts. Gap junctions were visualized immunohistochemically with an antibody raised against a unique cytoplasmic epitope of connexin43, and the spatial distribution pattern was determined by three-dimensional reconstruction. The results demonstrate that from embryonic day 13 onward, connexin43 becomes detectable immunohistochemically in the myocardium of atria and ventricles. No expression is initially detectable in the myocardium of the sinus venosus, the sinoatrial node, the posterior wall of the atrium and pulmonary veins, the interatrial septum, the atrioventricular canal, including atrioventricular node and bundle, the interventricular septum, and the outflow tract. The developmental increase in the density of gap junctions in atria and ventricles of prenatal hearts correlates well with the reported developmental increase in conduction velocity. Whereas connexin43 becomes expressed in the derivatives of the sinus venosus (except for the sinoatrial node) and in the subepicardial layer of the ventricular free wall shortly before birth, it remains undetectable in the atrioventricular node and bundle and the proximal part of the ventricular conduction tissue, even in the adult heart. The apparent absence of an abundant expression of connexin43 at a location with a supposedly high conduction velocity (i.e., the atrioventricular bundle and bundle branches) is unexpected. These observations were confirmed in studies of the adult mouse heart, which showed, in addition, that connexin32 is not expressed in any part of the heart.

Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein.

Currents from gap junction channels were recorded from pairs of astrocytes in primary culture using the double whole-cell recording technique. In weakly coupled pairs, single-channel events could be resolved without pharmacological uncoupling treatment. Under these conditions, unitary conductance was 56 +/- 7 pS, and except for multiples of this value, no other level of conductance was observed consistently. To characterize the type of junctional protein constituting astrocyte gap junction channels, immunological and biochemical experiments were carried out on the same material. Specific cDNA probes for three connexins identified in mammals (Cx26, Cx32, and Cx43) showed that only Cx43 mRNA was expressed in cultured astrocytes. The presence of Cx43 protein in cultured astrocytes was demonstrated by immunoblotting, immunofluorescence, and immunogold labeling using anti-peptide antibodies specific to Cx43. These results strongly suggest that gap junctions in astrocytes have a 50-60 pS unitary conductance associated with channels composed of Cx43 protein.

Changes in the expression of connexin 43, a cardiac gap junctional protein, during mouse heart development.

A cDNA probe coding for rat connexin 43 (Beyer et al., 1987), a gap junctional protein, was used to detect specific mRNA and estimate its relative abundance in mouse heart at different developmental stages: 11, 14 and 19 days post-coïtum (dpc); 1, 2 and 3 weeks post-partum (wpp), and at the adult stage. On Northern blots of total cellular RNA, a single 3.0 kb message was detected at all stages of development, and the differential intensities of labeling indicated developmental changes in mRNA abundance. mRNA levels were further investigated by dot-blotting. Densitometric analyses of dot-blot autoradiograms showed a five-fold increase of the mRNA level between 11 dpc and 1 wpp, then a gradual decrease until the adult stage where it reached a value close to that detected at 11 dpc. By comparison, myosin heavy chains and glycerldehyde-3-phosphate dehydrogenase mRNAs were found to peak at 3 wpp and 14 dpc, respectively. The presence and the relative abundance of connexin 43 were investigated at the same developmental stages as previously by immunoblotting of whole-ventricle fractions using antipeptide antibodies specific for this junctional protein. Quantitative data obtained from densitometric analyses of immunoblots showed that from 14 dpc to 1 wpp intensity of labeling of connexin 43 was roughly multiplied by a factor of 10. It peaked at 3 wpp before dropping to about 20% at the adult stage. The data obtained with both the cDNA probe and the antibodies were significant as shown by variance analyses. They suggest that expression of cardiac connexin 43 is developmentally-regulated: at the early stages of heart development the expression levels of the protein would seem to be mainly regulated by mRNA abundance; beyond 2 weeks after birth, the levels of connexin 43 would seem rather to depend upon its stability and/or the efficiency of the translation.

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein, in mammal heart and brain.

According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314-322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrahin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions. The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43. Proteins of Mr 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.

Cross-linking of cardiac gap junction connexons by thiol/disulfide exchanges.

SDS-polyacrylamide gel electrophoresis and immunoblotting were used to investigate inter- and intramolecular disulfide bonds to connexin 43 (the cardiac gap junctional protein) in isolated rat heart gap junctions and in whole heart fractions. In gap junctions isolated in the absence of alkylating agent, connexin 43 molecules are cross-linked by disulfide bonds. The use of iodoacetamide (100 mM) for the first steps of isolation procedure prevents the formation of these artifactual linkages. Investigation of connexin 43 in whole heart fractions by means of antibodies confirms the results obtained with isolated gap junctions; that is, connexin 43 molecules are not interconnected with disulfide bridges. In whole heart fractions treated with alkylating agents, a 38 kD protein, immunologically related to connexin 43, and containing intramolecular disulfide bonds is detected. It is hypothesized that this protein might be a folded form of connexin 43, a precursory form of the molecules embedded in the gap junctions.