In this study we show that androgens control the expression level of Cx32—and hence the extent of gap junction formation—post-translationally in androgen-responsive human prostate cancer cells in which the assembly of Cxs into gap junctions is highly efficient. In the absence of androgens, a major fraction of Cx32 is degraded from ER, presumably by ERAD, whereas in their presence, this fraction is rescued from degradation. Thus, androgens seem to regulate the formation and degradation of gap junctions in a novel way: by rerouting the pool of Cx32, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing the cell surface–associated pool amenable for gap junction assembly. Moreover, we also show that Cx32 and Cx43 degrade by a similar mechanism and that degradation does not involve the entire cytoplasmic tail of Cx32. The significance of these data are further underscored by the lack of appreciable effect of androgens on the degradation of adherens junction–associated proteins, E-cadherin and α- and β-catenins, and tight junction–associated protein, occludin. Thus, in this model system, androgen-regulated proteasome—and not the lysosome—mediated degradation of Cx32 appeared to be the major control point in modulating the formation and degradation of gap junctions.
The present study was prompted by three independent lines of inquiry. First, previous findings had shown that Cx32 was expressed by the well-differentiated cells of the liver (Dermietzel et al., 1987
; Stutenkemper et al., 1992
; Kojima et al., 1996
) and cells derived from other hormone-responsive tissues, such as pancreas and thyroid, where gap junctional communication had been documented to play an important role in regulating differentiation (Munari-Sliem et al., 1991
; Munari-Sliem and Rousset, 1996
; Meda, 1996
; Guerrier et al., 1995
; Stock et al., 1998
). Second, we had previously shown that Cx32 was expressed by the well-differentiated epithelial cells of the prostate and prostate tumors and that its expression coincided with the morphological, histological and cellular (secretory) differentiation of the prostate (Mehta et al., 1999
; Habermann et al., 2001
). Third, consistent with the tumor suppressing and prodifferentiating role of Cx32 in vivo and in vitro (Mehta et al., 1999
; King and Lampe, 2004
; King et al., 2005
), and on the basis of the prodifferentiating role of androgens in prostate morphogenesis (Cunha et al., 1992
), we reasoned that androgens might regulate its assembly into gap junctions.
Multiple lines of evidence suggested that in the absence of androgens a major proportion of Cx32 was degraded from the ER en route to the Golgi in a proteasomal inhibitor-sensitive manner. First, in the absence of androgens, Cx32 was barely detectable by immunocytochemical analysis at 37°C, or at 15°C, when its transport from ER to Golgi was blocked (B). Second, inhibition of proteasomal function at 15°C under androgen-depleted conditions enhanced expression level of Cx32 and caused its intracellular accumulation (B). Third, ER exit of Cx32 and its subsequent accumulation in the cis-Golgi upon monensin block was observed only when cells were grown in androgen-containing medium (A). Fourth, a DsRed-Cx32 chimera that remained trapped in the ER was also degraded upon androgen depletion and its degradation was prevented by MG132 (A). Fifth, pulse chase analysis of metabolically labeled Cx32 indicated that it degraded rapidly soon after its synthesis in cells grown in charcoal-stripped (androgen-depleted) serum in stark contrast to cells grown in normal serum and in charcoal-stripped serum supplemented with the androgen or lactacystin or ALLN. Finally, inhibiting lysosomal function under androgen-depleted conditions prevented the degradation of only cell surface and gap junction-associated pool of Cx32 (). These results suggest that in the absence of androgens, Cx32 is degraded soon after its synthesis and that only the newly synthesized intracellular pool of Cx32 appears to be affected by the androgens or MG132 under androgen-depleted conditions. The gap junctional plaque associated pool seems to be predominantly degraded in the lysosome both in the presence and absence of androgens. On inhibition of proteasomal function, intracellularly accumulated Cx32 appeared to localize in discrete puncta or aggregates of variable sizes that were found to be scattered through out the cytoplasm. These puncta did not seem to reside in the ER, Golgi, and trans-Golgi network, as assessed by colocalization studies using well-established markers for these organelles and seemed to reside in as yet undefined compartment as assessed by the cell fractionation assay (C). Further studies are required to identify the biochemical nature of these aggregates and the subcellular compartments in which they reside.
As assessed by quantitative real-time PCR analysis, androgens neither induced the transcription of endogenous Cx32 gene (data not shown) nor affected Cx32 mRNA level driven by the retroviral promoter (D). Moreover, it also seemed unlikely that androgens regulated the expression of Cx32 at the translational level, because inhibition of proteasomal function under androgen-depleted conditions increased steady state expression level and caused substantial intracellular accumulation (A). These data indicate that in the absence of androgens, Cx32 protein was synthesized but was degraded immediately after its synthesis. Furthermore, the fact that Cx32 accumulated intracellularly to a similar extent upon inhibition of proteasomal function both in the presence and absence of androgens at 15°C, when ER-to-Golgi transport was blocked (Bannykh et al., 1996
; Hauri et al., 2000
), argue further against translational control as one of the mechanisms for decreasing the expression level (B). Thus, the level of Cx32 seemed to be controlled by the androgens post-translationally. Because Cx43 was also degraded upon androgen depletion, it appears that this may be a common mechanism by which expression level of some Cxs is regulated to permit gap junction assembly. In previous findings a decrease in, or loss of, Cx32 expression was observed upon partial hepatectomy and in primary cultures of rat hepatocytes, without significant changes in the mRNA level, but no rational explanation was put forward (Dermietzel et al., 1987
; Kren et al., 1993
; Kojima et al., 1996
). It is possible that in those studies the expression level of Cx32 was regulated post-translationally by a similar mechanism involving degradation.
Our findings suggest that AR-mediated signaling was required for maintaining Cx32 expression level and preventing its degradation post-translationally. In AR-negative DU-145 cells, in which Cx32 was introduced using recombinant retrovirus, depletion of androgens neither enhanced its expression level nor induced degradation. Moreover, in androgen-responsive LNCaP cells, Cx32 degraded rapidly in androgen-containing medium upon addition of an anti-androgen, bicalutamide, which inhibits AR-mediated signaling by competing with the androgens (Iversen, 2002
). These findings document that the depletion of androgens is the predominant factor responsible for the degradation of Cx32 when cells are grown in charcoal-stripped serum—and not other factors that might also be removed upon charcoal stripping. When AR was overexpressed in Cx32-expressing LNCaP cells, the expression level of Cx32 was increased, along with the size of the gap junctions, most likely due to increased level of AR which was available for the hormones to bind. These results also corroborate the androgen dose–response data and concur with the notion that androgens themselves increase AR level by stabilizing it (Dehm and Tindall, 2005
; Shen and Coetzee, 2005
One salient feature of our data is that androgen depletion induced degradation of only Cx32 and had no effect on the degradation of adherens junction–associated proteins E-cadherin and α- and β-catenins and tight junction–associated protein, occludin (). Because cadherins had previously been shown to facilitate the trafficking and assembly of Cxs into gap junctions (Musil et al., 1990
; Jongen et al., 1991
; Hernandez-Blazquez et al., 2001
), these data imply that the degradation of Cx32 and gap junctions composed of it was not triggered indirectly by the loss of E-cadherin or disassembly of adherens junctions upon removal of androgens. It is worth noticing that although the total level of occludin did not decrease, androgen depletion decreased its detergent-insoluble fraction, with a concomitant increase in the soluble fraction, causing it to accumulate intracellularly. One possible interpretation of these data are that the trafficking of occludin to the cell surface and its detergent insolubility (that is, incorporation into tight junctions) might be regulated by Cx32. This line of thought is well supported by earlier studies, which showed that in cell lines derived from Cx32 knock out mice, occludin remained in the cytoplasm and reintroduction of Cx32 induced its trafficking and assembly into tight junctions (Kojima et al., 2001
). Thus, it is possible that an increase in the detergent solubility of occludin is indirectly affected by the degradation of Cx32 and gap junctions upon androgen removal. Further studies are required to substantiate this notion.
Although Cx32 and Cx43 have previously been shown to be degraded by the proteasomal and the lysosomal pathways in several cell culture model systems (Laing and Beyer, 1995
; Laing et al., 1997
; Musil et al., 2000
; VanSlyke and Musil, 2002
; Laird, 2005
; VanSlyke and Musil, 2005
), the physiological stimuli that trigger their degradation or dictate the choice of the pathway have not yet been explored. Using cell lines that differed widely in their ability to assemble Cx43 and Cx32 into functional gap junctions, a substantial portion of newly synthesized Cx43 and Cx32 was shown to be degraded by ERAD; moreover, the degradation was prevented by nontoxic hyperthermic and oxidative stress or by proteasomal inhibitors and was accompanied by enhanced gap junction assembly and function (Musil et al., 2000
; VanSlyke and Musil, 2005
). Furthermore, similar treatments also increased gap junctions by preventing the degradation of the cell surface–associated pool of Cx43 and by enhancing its recycling (VanSlyke and Musil, 2005
). Thus, it has been proposed that regulation of Cx turnover might be an important mechanism to enhance gap junction assembly and function upon physiological demand (Musil et al., 2000
; VanSlyke and Musil, 2005
). Our findings—that androgens enhanced the expression level of Cx32 by rerouting its presumably ERAD-targeted pool to the cell surface—lend credence to the above notion, and for the first time have identified a novel, hormonally regulated route by which the formation and degradation of gap junctions could be rapidly controlled in response to physiological stimuli from the ER. Although ERAD has been shown to be a common mechanism by which misfolded proteins are degraded, the molecular mechanisms have not yet been elucidated (Arvan et al., 2002
; Ellgaard and Helenius, 2003
; McCracken and Brodsky, 2003
; Meusser et al., 2005
). Hence, the mechanism by which androgens reroute the ERAD-targeted pool of Cx32 to the cell surface is likely to be complex and may be related to induction and repression of factors that regulate Cx trafficking and degradation or both. Based on our findings, it is tempting to speculate that degradation of Cxs before their assembly into gap junctions by ERAD itself is a physiologically regulated event.
Requirement for androgens throughout normal prostatic development and prostate cancer progression is well established (Cunha et al., 1987
). Androgen-responsive LNCaP cells have been widely used for studying the progression of human prostate cancer from an androgen-dependent state to an androgen-independent state in vitro (Webber et al., 1996
). Although the molecular events involved during the progression of prostate cancer from an indolent, hormone (androgen)-dependent state to an invasive, androgen-independent state are not fully understood, the progression is characterized by the emergence of cells that no longer depend on androgens for survival (Abate-Shen and Shen, 2000
; Feldman and Feldman, 2001
). Because rescue of Cx32 and Cx43 degradation upon addition of androgens was accompanied by parallel changes in gap junction function, our findings suggest that it is likely to be intimately involved in regulating the physiological functions of the prostate. Moreover, the findings that in a cell culture model that mimics the progression of human prostate cancer (Igawa et al., 2002
), degradation of Cxs as well as formation of gap junctions—and not of other junction-associated proteins—is androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.