Altered intercellular communication in lung fibroblast cultures from patients with idiopathic pulmonary fibrosis

RATIONALE: Gap junctions are membrane channels formed by an array of connexins which links adjacent cells realizing an electro- metabolic synapse. Connexin-mediated communication is crucial in the regulation of cell growth, differentiation, and development. The activation and proliferation of phenotypically altered fibroblasts are central events in the pathogenesis of idiopathic pulmonary fibrosis. We sought to evaluate the role of connexin-43, the most abundant gap-junction subunit in the human lung, in the pathogenesis of this condition. METHODS: We investigated the transcription and protein expression of connexin-43 and the gap-junctional intercellular communication (GJIC) in 5 primary lung fibroblast lines derived from normal subjects (NF) and from 3 histologically proven IPF patients (FF). RESULTS: Here we show that connexin-43 mRNA was significantly reduced in FF as demonstrated by standard and quantitative RT-PCR. GJIC was functionally evaluated by means of flow-cytometry. In order to demonstrate that dye spreading was taking place through gap junctions, we used carbenoxolone as a pharmacological gap-junction blocker. Carbenoxolone specifically blocked GJIC in our system in a concentration dependent manner. FF showed a significantly reduced homologous GJIC compared to NF. Similarly, GJIC was significantly impaired in FF when a heterologous NF line was used as dye donor, suggesting a complete defect in GJIC of FF. CONCLUSION: These results suggest a novel alteration in primary lung fibroblasts from IPF patients. The reduced Cx43 expression and the associated alteration in cell-to-cell communication may justify some of the known pathological characteristic of this devastating disease that still represents a challenge to the medical practice

Assembly of Connexin43 into Gap Junctions Is Regulated Differentially by E-Cadherin and N-Cadherin in Rat Liver Epithelial Cells

E-cadherin and N-cadherin affect the assembly of connexin43 into gap junctions differentially. N-cadherin disrupts assembly by triggering endocytosis of connexin43, whereas E-cadherin facilitates the assembly

Regulatory Effect of Connexin 43 on Basal Ca2+ Signaling in Rat Ventricular Myocytes

Background: It has been found that gap junction-associated intracellular Ca 2+ [Ca 2+]i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca 2+ signaling, in particular the basal [Ca 2+] i activities, is unclear. Methods and Results: Global and local Ca 2+ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca 2+ transients and local Ca 2+ sparks in monolayer NRVMs and Ca 2+ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP3 butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca 2+ signal and LY uptake by gap uncouplers, whereas blockade of IP 3 receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca 2+ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibod

Retinoids Regulate the Formation and Degradation of Gap Junctions in Androgen-Responsive Human Prostate Cancer Cells

The retinoids, the natural or synthetic derivatives of Vitamin A (retinol), are essential for the normal development of prostate and have been shown to modulate prostate cancer progression in vivo as well as to modulate growth of several prostate cancer cell lines. 9-cis-retinoic acid and all-trans-retinoic acid are the two most important metabolites of retinol. Gap junctions, formed of proteins called connexins, are ensembles of intercellular channels that permit the exchange of small growth regulatory molecules between adjoining cells. Gap junctional communication is instrumental in the control of cell growth. We examined the effect of 9-cis-retinoic acid and all-trans retinoic acid on the formation and degradation of gap junctions as well as on junctional communication in an androgen-responsive prostate cancer cell line, LNCaP, which expressed retrovirally introduced connexin32, a connexin expressed by the luminal cells and well-differentiated cells of prostate tumors. Our results showed that 9-cis-retinoic acid and all-trans retinoic acid enhanced the assembly of connexin32 into gap junctions. Our results further showed that 9-cis-retinoic acid and all-trans-retinoic acid prevented androgen-regulated degradation of gap junctions, post-translationally, independent of androgen receptor mediated signaling. Finally, our findings showed that formation of gap junctions sensitized connexin32-expressing LNCaP cells to the growth modifying effects of 9-cis-retinoic acid, all-trans-retinoic acid and androgens. Thus, the effects of retinoids and androgens on growth and the formation and degradation of gap junctions and their function might be related to their ability to modulate prostate growth and cancer

Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.

Activation of furin requires autoproteolytic cleavage of its 83-amino acid propeptide at the consensus furin site, Arg-Thr-Lys-Arg107/. This RER-localized cleavage is necessary, but not sufficient, for enzyme activation. Rather, full activation of furin requires exposure to, and correct routing within, the TGN/endosomal system. Here, we identify the steps in addition to the initial propeptide cleavage necessary for activation of furin. Exposure of membrane preparations containing an inactive RER-localized soluble furin construct to either: (i) an acidic and calcium-containing environment characteristic of the TGN; or (ii) mild trypsinization at neutral pH, resulted in the activation of the endoprotease. Taken together, these results suggest that the pH drop facilitates the removal of a furin inhibitor. Consistent with these findings, following cleavage in the RER, the furin propeptide remains associated with the enzyme and functions as a potent inhibitor of the endoprotease. Co-immunoprecipitation studies coupled with analysis by mass spectrometry show that release of the propeptide at acidic pH, and hence activation of furin, requires a second cleavage within the autoinhibitory domain at a site containing a P6 arginine (-Arg70-Gly-Val-Thr-Lys-Arg75/-). The significance of this cleavage in regulating the compartment-specific activation of furin, and the relationship of the furin activation pathway to those of other serine endoproteases are discussed

The Major Core Protein P4a (A10L Gene) of Vaccinia Virus Is Essential for Correct Assembly of Viral DNA into the Nucleoprotein Complex To Form Immature Viral Particles

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-β-d-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles