Apical and basolateral EGF receptors regulate gastric mucosal paracellular permeability.

Previous studies found that monolayers formed from canine oxyntic epithelial cells in primary culture displayed remarkable resistance to apical acidification and both mitogenic and migratory responses to epidermal growth factor (EGF) treatment. In our present studies, we found that EGF increased transepithelial resistance (TER) but not short-circuit current in these monolayers. Parallel effects of EGF on decreasing mannitol flux and increasing TER implicate direct regulation of paracellular permeability. EGF acting at either apical and basolateral receptors rapidly increased TER, but the apical response was sustained whereas the basolateral response was transient. (125)I-labeled EGF binding revealed specific apical binding, but receptor numbers were 25-fold lower than on the basolateral surface. Both apical and basolateral EGF activated tyrosine phosphorylation of EGF receptors (EGFR), beta-catenin, and cellular substrate as evident on confocal microscopy. Although apical EGF activated a lesser degree of receptor autophosphorylation than basolateral EGF, phosphorylation of beta-catenin was equally prominent with apical and basolateral receptor activation. Together, these findings indicate that functional apical and basolateral EGFR exist on primary canine gastric epithelial cells and that these receptors regulate paracellular permeability. The sustained effect of apical EGFR activation and prominent phosphorylation of beta-catenin suggest that apical EGFR may play a key role in this regulation.

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture.

Transduction mechanisms between target cells within the intestinal wall and peripheral terminals of extrinsic primary afferent neurons are poorly understood. The purpose of this study was to characterize the interactions between smooth muscle cells from the rat distal colon and lumbar dorsal root ganglion (DRG) neurons in coculture. DRG neurons visually appeared to make contact with several myocytes. We show that brief mechanical stimulation of these myocytes resulted in intracellular Ca2+ concentration ([Ca2+]i) transients that propagated into 57% of the contacting neurites. Direct mechanical stimulation of DRG neurites cultured without smooth muscle had no effect. We also show that colonic smooth muscle cells express multiple connexin mRNAs and that these connexins formed functional gap junctions, as evidenced by the intercellular transfer of Lucifer yellow. Furthermore, thapsigargin pretreatment and neuronal heparin injection abolished the increase in neurite [Ca2+]i, indicating that the neuronal Ca2+ signal was triggered by inositol 1,4, 5-trisphosphate-mediated Ca2+ release from intracellular stores. Our results provide evidence for intercellular chemical communication between DRG neurites and intestinal smooth muscle cells that mediates the exchange of second messenger molecules between different cell types.

Dominant inhibition of intercellular communication by two chimeric connexins.

1. The physiological significance of communication through gap junction channels has been difficult to assess because channel activity cannot be experimentally modulated in a specific manner. To address this problem we have constructed chimeric connexins that function as dominant-negative inhibitors of intercellular channel activity.

Connexins, connexons, and intercellular communication.

Cells in tissues share ions, second messengers, and small metabolites through clusters of intercellular channels called gap junctions. This type of intercellular communication permits coordinated cellular activity. Intercellular channels are formed from two oligomeric integral membrane protein assemblies, called connexons, which span two adjacent cells' plasma membranes and join in a narrow, extracellular "gap." Connexons are formed from connexins, a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeability of the intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter-connexin interactions and uncovered novel characteristics of the channel permeability and gating behavior. Structure-function studies provide a molecular understanding of the significance of connexin diversity and demonstrate the unique regulation of connexins by tyrosine kinases and oncogenes.

Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication.

We show that connexin expression and in vivo patterns of communication were dramatically altered in response to epidermal wounding. Six hours after injury, Cx26 was up-regulated in the differentiated cells proximal to the wound, but was down-regulated in cells located at the wound edge. In contrast, Cx31.1 and Cx43 were down-regulated in cells both peripheral to and at the wounded edge. These patterns of altered connexin expression were detectable as early as 2 h after wounding and were most pronounced in 24-h old wounds. Increased expression of Cx26 was still evident in the hyperproliferative epidermis of 6-day old wounds. In vivo dye transfer experiments with Lucifer yellow and neurobiotin confirmed that junctional communication patterns were altered in ways consistent with changes in connexin expression. The data thus suggest that intercellular communication is intimately involved in regulating epidermal wound repair.

Expression of gap junction proteins Cx26, Cx31.1, Cx37, and Cx43 in developing and mature rat epidermis.

To elucidate mechanisms underlying gap junction-mediated intercellular communication in epidermal keratinocytes, we have examined the expression of four connexin genes, Cx26, Cx31.1, Cx37, and Cx43, in fetal (embryonic day 17-18), newborn (post-natal day 0), and mature rat skin. Northern analyses of total skin RNA showed that levels of Cx26, Cx37, and Cx43 mRNAs remained relatively constant throughout the three developmental stages examined, whereas Cx31.1 mRNA was 15-30 times more abundant in mature skin than in fetal skin. Antibodies specifically recognizing these connexin proteins were then used in conjunction with a recently described amplification technique to immunohistochemically stain sections of paraffin embedded rat tail epidermis. We show that Cx26, Cx31.1, Cx37, and Cx43 display overlapping but distinct patterns of expression within the keratinocyte cell layers of developing and mature epidermis.

Mouse F9 teratocarcinoma stem cells expressing the stably transfected homeobox gene Hox 1.6 exhibit an altered morphology.

Expression of the murine homeobox gene Hox 1.6 rapidly increases in F9 teratocarcinoma cells when these cells are induced with retinoic acid to differentiate into primitive and parietal endoderm. Hox 1.6 encodes a putative transcriptional regulatory protein which may function as a secondary regulator of gene expression during the differentiation process. To examine the role of the Hox 1.6 gene, we have stably transfected F9 stem cells with a cDNA containing the complete coding sequence of Hox 1.6 under the control of the mouse metallothionein promoter. Two clonally distinct cell lines that express high levels of the transfected Hox 1.6 gene have been isolated and characterized. We show that expression of the transfected Hox 1.6 gene in F9 cells dramatically alters the stem cell morphology. However, the transfected cells do not differentiate in the absence of retinoic acid treatment, nor are they prevented from differentiating in response to such treatments. We therefore suggest that the Hox 1.6 gene controls the expression of genes which influence changes in F9 cell morphology during RA-induced differentiation.

Specificity and mechanism of antitermination by Q proteins of bacteriophages lambda and 82.

Lambdoid phage late gene operons are positively regulated by genome-specific antiterminator proteins encoded by the Q gene of each phage. In this paper, we compare the activity of phage lambda and phage 82 Q proteins. Q82-mediated antitermination, like that of Q lambda, involves a transcription pause during which the regulator can modify RNA polymerase. We show that the activities of both Q82 and Q lambda are genome-specific in chasing RNA polymerase out of the early pause sites and in mediating antitermination. Finally, we show that the length of the RNA in the paused complex, or the exact position of the pause, affects the efficiency with which Q82 chases RNA polymerase out of the pause.

Sequences required for antitermination by phage 82 Q protein.

The gene Q antiterminator proteins of phages lambda and 82 modify RNA polymerase at sites (named qut) that are close to, and apparently inseparable from the promoters themselves. Modification occurs while RNA polymerase has paused close to the start site, at nucleotide 16 for lambda, and nucleotides 15 and 25 for phage 82. We present a deletion analysis of the phage 82 qut site that identifies sequences required for pausing and shows that these sequences also are required for efficient Q function in vivo and in vitro. We show (1) that deletions as close as +5 to the RNA start site retain some ability to be modified by Q82, suggesting that part of the qut site is in the non-transcribed region of the promoter; (2) that NusA protein is required for activity of Q82 on certain qut82 site deletions, whereas it only modestly stimulates antitermination from the native qut82 site; and (3) that qut82 is active only on RNA polymerase that initiates at the qut-associated promoter, and not on RNA polymerase that initiates upstream and passes through an otherwise active qut82 site.

Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase.

We have constructed novel transcription templates in which we have fused the late gene promoters of Escherichia coli phages lambda and 82 upstream from three different rho-independent transcription terminators. Using an in vitro transcription assay and an in vivo galactokinase expression assay, we find that the initial portion of the transcribed region significantly affects the efficiency of some downstream terminators. We have identified, by deletion, substitution and point mutation analysis, sequences responsible for these increased levels of factor-independent readthrough. Since these important sequences occur within about 30 nucleotides of the RNA start site, we suggest that the initial portion of the transcript can affect termination efficiency.

Bacteriophage 82 gene Q and Q protein. Sequence, overproduction, and activity as a transcription antiterminator in vitro.

Phage 82 gene Q encodes a phage-specific positive regulator of late gene expression, thought, by analogy to the corresponding gene of phage lambda, to be a transcription antiterminator. We have cloned and sequenced the phage 82 gene Q and have overproduced and purified the 82 Q protein. We also have identified and sequenced DNA containing the phage 82 late gene promoter and terminator. We show that purified 82 Q protein is active and specific for DNA containing the 82 late gene promoter in a well defined in vitro transcription reaction: RNA polymerase initiating at the phage 82 late gene promoter and modified by 82 Q protein reads through a downstream transcriptional terminator. We used T1 RNase mapping to confirm that the putative readthrough RNA made in the presence of 82 Q protein is in fact an elongation product of the shorter RNA.