Connexin32: Sorting, Endocytosis and Assembly

Gap junctions (GJ)s are conglomeration of several cell-cell channels at cell-to-cell contact sites involved in the direct intercellular exchange of small growth regulatory molecules. Defects in assembly of GJ-forming proteins, called connexins (Cxs), are observed in many cancers, yet the molecular basis of this defect remains unknown. Connexin32 (Cx32) is expressed by the polarized cells in epithelia. The carboxyl-terminal tail (CT) of Cx32, although not required to initiate GJ formation, orchestrates several aspects of GJ dynamics, function and growth. Our studies have discovered that the CT of Cx32 harbors a tyrosine-based [YXXØ]-type and two dileucine-based [DE]XXXL[LI]-type motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins. We explored their role in regulating endocytosis and GJ-forming ability of Cx32. One dileucine motif, designated as LI, was located near the juxtamembrane domain, whereas another, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR. Our results showed that the LL and LR motifs regulated the endocytosis of Cx32 by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited GJ assembly by augmenting Cx32 endocytosis via the LL and LR motifs. We also found that the tyrosine-based motif, acted as a possible endocytic motif. Moreover, our studies showed that the LI motif regulates basolateral sorting of Cx32 from the Golgi to the cell surface in polarized cells only. We also found that the CT of Cx32 harbored three cysteines. These cysteines were likely to be modified by palmitoylation, and two of them were part of a CAAX box motif, known to be modified by prenylation. Our studies have shown for the first time that cysteine 217 of Cx32 was palmitoylated. However, we found that mutating these cysteines singly affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, we discovered that mutating all cysteines together or cysteine 280 and 283 in combination, blocked the transport of Cx32 from the Golgi to the cell surface. Overall, our studies here show that the trafficking, endocytosis, and assembly of Cx32 are modulated in a complex manner by different regulatory motifs found on its CT

Saturated free fatty acids induce cholangiocyte lipoapoptosis

Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. CONCLUSION: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients

Vitamin D3 regulates the formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

1α-25(OH)2 vitamin D3 (1-25D), an active hormonal form of Vitamin D3, is a well-known chemopreventive and pro-differentiating agent. It has been shown to inhibit the growth of several prostate cancer cell lines. Gap junctions, formed of proteins called connexins (Cx), are ensembles of cell-cell channels, which permit the exchange of small growth regulatory molecules between adjoining cells. Cell-cell communication mediated by gap junctional channels is an important homeostatic control mechanism for regulating cell growth and differentiation. We have investigated the effect of 1-25D on the formation and degradation of gap junctions in an androgen-responsive prostate cancer cell line, LNCaP, which expresses retrovirally-introduced Cx32. Connexin32 is expressed by the luminal and well-differentiated cells of normal prostate and prostate tumors. Our results document that 1-25D enhances the expression of Cx32 and its subsequent assembly into gap junctions. Our results further show that 1-25D prevents androgen-regulated degradation of Cx32, post-translationally, independent of androgen receptor (AR)-mediated signaling. Finally, our findings document that formation of gap junctions sensitizes Cx32-expressing LNCaP cells to the growth inhibitory effects of 1-25D and alters their morphology. These findings suggest that the growth-inhibitory effects of 1-25D in LNCaP cells may be related to its ability to modulate the assembly of Cx32 into gap junctions

Cx32 expression sensitizes LNCaP cells to growth inhibitory effect of 1-25D.

<p>LNCaP-P, LNCaP-N and LNCaP-32 cells were seeded in 6-cm dishes in replicate (5×10⁴ cells/dish) and treated with 1-25D (1 nM or 2.5 nM) and MB (2.5 nM). Cells were grown for 10 days with a medium change at day 2 and 5. Cells were trypsinized and counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106437#s2" target="_blank">Materials and Methods</a>. The values represent Mean number of cells per dish ×10⁵ ± SE of the Mean. Values in the parentheses represent Means of % Growth ± SE of the Mean.</p>a<p>P≥0.12.</p>b<p>P≤0.05.</p><p>Cx32 expression sensitizes LNCaP cells to growth inhibitory effect of 1-25D.</p

Effect of 1-25D and androgens on junctional transfer of fluorescent tracers in LNCaP-32 cells under androgen-depleted conditions.

<p>LNCaP-32 cells were seeded as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106437#pone-0106437-t001" target="_blank">Table 1</a> legend. Cells were switched to charcoal-stripped, androgen-depleted medium (Strip) for 48 h in the presence and absence of 1-25D and synthetic (MB) and the natural (DHT) androgens.</p>a<p>The number of fluorescent cell neighbors (mean ± SE) 1 min (Lucifer Yellow), 3 min (Alexa-488) and 15 min (Alexa-594) after microinjection into test cell. The total number of injection trials is shown in parentheses.</p>b<p>Cells were stripped for 24 h in the presence and absence of 1-25D, DHT (10 nM) and MB (2.5 nM).</p>c<p>P≤0.0001 for normal serum (NS) versus stripped (Strip) for all tracers. A two tailed Student's <i>t</i> test was used to calculate P value assuming unequal variance.</p>d<p>P≤0.0001 for stripped serum (Strip) versus stripped and treated cells for all the tracers. A two tailed Student's <i>t</i> test was used to calculate P value assuming unequal variance.</p><p>Effect of 1-25D and androgens on junctional transfer of fluorescent tracers in LNCaP-32 cells under androgen-depleted conditions.</p

The effect of combined treatment of 1-25D with androgens and retinoids on the expression level of Cx32 and the adherens-junction-associated proteins.

<p>Cx32-expressing LNCaP-32 cells were treated with the 1-25D, 9-CRA, DHT and MB as indicated. <b>A</b>. Combined treatment with 1-25D with MB or 9-CRA is more effective in increasing Cx32 expression level than treatment with the single agent alone. <b>B</b>. Quantitative analysis of the expression level of the data shown in <b>A</b>. Each bar represents the Mean and the Standard Error of the Mean from 4-13 experiments. The asterisks (**) indicate P value of ≤0.0001. A two tailed Student's <i>t</i> test was used to calculate P value assuming unequal variance. <b>C</b>. Effect of 1-25D on adherens junction associated proteins. Expression of adherens junction proteins E-cadherin (E-cad), α-catenin (α-cat), and β-catenin (β-cat) was analyzed by Western blot analysis of total cell lysate (10 µg). Note that there is no effect.</p

Effect of 1,25D and androgen on the junctional transfer of fluorescent tracers in LNCaP-32 cells.

<p>LNCaP-32 cells, seeded in 6 cm dishes in replicate, were grown to 65–70% confluence. Junctional transfer was measured after microinjecting fluorescent tracers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106437#s2" target="_blank">Materials and Methods</a>).</p>a<p>The number of fluorescent cell neighbors (Mean ± SE) 1 min (Lucifer Yellow), 3 min (Alexa-488) and 15 min (Alexa-594) after microinjection into test cell. The total number of injection trials is shown in parentheses.</p>b<p>Cells were treated for 48 h with 1-25D, DHT (10 nM) and MB (2.5 nM).</p>c<p>P≤0.0001 for normal serum versus stripped and treated cells. A two tailed Student's <i>t</i> test was used to calculate P value assuming unequal variance.</p><p>Effect of 1,25D and androgen on the junctional transfer of fluorescent tracers in LNCaP-32 cells.</p

1-25D alters the morphological phenotype of Cx32-expressing LNCaP cells.

<p>LNCaP-P, LNCaP-N and LNCaP-32 cells were seeded at a density of 2-3×10⁴ per 6-cm dish and treated with the indicated concentrations of 1-25D after 24 hrs. After 5 days, cells were fixed and stained with crystal violet. Note robust morphological changes in LNCaP-32 cells treated with 1-25D.</p

Effect of 1-25D and MB on the formation of gap junctions in the presence and the absence of Casodex.

<p>LNCaP-32 cells, seeded on glass cover slips, were grown to 70% confluence. Cells were then grown for additional 24 h in normal medium (NS), androgen-depleted medium alone (ST), normal serum supplemented with Casodex (CDX; NS+CDX), androgen-depleted medium supplemented with MB (ST+MB), MB and Casodex (ST+MB+CDX), 1-25D (ST+1-25D), 1-25D and Casodex (ST+1-25D+CDX) and in normal serum with 1-25D (NS+1-25D). Gap junction formation was assessed immunocytochemically. Note that GJs (green) are not degraded in cells treated with 1-25D both in the presence and absence of Casodex whereas they are degraded in normal serum and androgen-depleted but MB supplemented medium containing Casodex. E-cad is shown in red and the nuclei (blue) are stained with DAPI. Scale Bar = 20 µM.</p