Cx30.2 deletion causes imbalances in testicular Cx43, Cx46, and Cx50 and insulin receptors. Reciprocally, diabetes/obesity alters Cx30.2 in mouse testis.

Cx30.2 protein content and localization were assessed during development. An account of Cx30.2, Cx43, Cx46, and Cx50, and insulin receptor (IR) responses to Cx30.2, Cx46, or Cx50 deficiency in mouse interstitial tissue (ITf)- and seminiferous tubule-enriched fractions (STf) is given. The impact of high glucose/insulin on Cx30.2 was investigated in spontaneously diabetic and obese db/db and ob/ob mouse testis and anterior pituitary (AP). Cx30.2 labeled contacts in vascular endothelial and Leydig cells and Sertoli cell junctions in stage V-VII. Cx30.2 expression is regulated differently in the interstitium and tubules. Cx30.2 at 30-kDa levels peaked by 28 days in ITf and by 14 days in STf. In STf, deleting Cx30.2 decreased Cx43 and Cx50, whereas deleting Cx50 downregulated Cx30.2. The opposite occurred in ITf. In STf, deleting Cx30.2 upregulated Cx46 except the full-length reciprocally, deleting Cx46 upregulated Cx30.2. In ITf, Cx30.2 deficiency upregulated full-length and phosphorylated Cx46, whereas deleting Cx46 downregulated 48- to 50-kDa Cx30.2. The db/db and ob/ob mouse ITf, STf, and AP showed imbalanced Cx30.2 levels. IRα levels at 135 kDa declined in Cx30.2-/- and Cx50-/- mouse ITf and Cx46-/- and Cx50-/- STf. IRß at 98 to 110 kDa dropped in Cx30.2-/- and Cx46-/- mice STf suggesting that Cx30.2 deficiency decreases active IR sites. The results show the connexins interdependence and interaction and that altering a single connexin changes the remaining connexins expression, which can modify gap junction-mediated glucose exchanges in contacting cells. Data suggest that glucose/insulin influences Cx30.2 turnover in testis and AP and, reciprocally, that connexins modulate testis glucose uptake and response to insulin.

The anterior pituitary gap junctions: potential targets for toxicants.

The anterior pituitary regulates endocrine organs and physiological activities in the body. Environmental pollutants and drugs deleterious to the endocrine system may affect anterior pituitary activity through direct action on anterior pituitary cells. Within the gland, endocrine and folliculostellate cells are organized into and function as individual tridimensional networks, each network regulating its activity by coordinating the connected cells' responses to physiological or pathological cues. The gap junctions connecting endocrine cells and/or folliculostellate cells allow transmission of information among cells that is necessary for adequate network function. Toxicants may affect gap junctions as well as the physiology of the anterior pituitary. However, whether toxicants effects on anterior pituitary hormone secretion involve gap junctions is unknown. The folliculostellate cell gap junctions are sensitive to hormones, cytokines and growth factors. These cells may be an interesting experimental model for evaluating whether toxicants target anterior pituitary gap junctions.

Cholesterol metabolism and Cx43, Cx46, and Cx50 gap junction protein expression and localization in normal and diabetic and obese ob/ob and db/db mouse testes.

Decreased fertility and birth rates arise from metabolic disorders. This study assesses cholesterol metabolism and Cx46, Cx50, and Cx43 expression in interstitium- and seminiferous tubule-enriched fractions of leptin-deficient ( ob/ob) and leptin receptor-deficient ( db/db) mice, two type 2 diabetes and obesity models associated with infertility. Testosterone levels decreased and glucose and free and esterified cholesterol (FC and EC) levels increased in serum, whereas FC and EC levels decreased in the interstitium, in ob/ob and db/db mice. In tubules, a decrease in EC caused FC-to-EC ratios to increase in db/db mice. In tubules, only acyl coenzyme A:cholesterol acyl transferase type 1 and 2 protein levels significantly decreased in ob/ob, but not db/db, mice compared with wild-type mice, and imbalances in the cholesterol transporters Niemann-Pick C1 (NPC1), ATP-binding cassette A1 (ABCA1), scavenger receptor class B member I (SR-BI), and cluster of differentiation 36 (CD36) were observed in ob/ob and db/db mice. In tubules, 14-kDa Cx46 prevailed during development, 48- to 49- and 68- to 71-kDa Cx46 prevailed during adulthood, and total Cx46 changed little. Compared with wild-type mice, 14-kDa Cx46 increased, whereas 48- to 49- and 68- to 71-kDa Cx46 decreased, in tubules, whereas the opposite occurred in the interstitium, in db/db and ob/ob mice. Total and 51-kDa Cx50 increased in db/db and ob/ob interstitium and tubules. Cx43 levels decreased in ob/ob interstitium and tubules, whereas Cx43 decreased in db/db interstitium but increased in db/db tubules. Apoptosis levels measured by ELISA and numbers of apostain-labeled apoptotic cells significantly increased in db/db, but not ob/ob, tubules. Testicular db/db capillaries were Cx50-positive but weakly Cx43-positive with a thickened lamina, suggesting altered permeability. Our findings indicate that the db mutation-induced impairment of meiosis may arise from imbalances in cholesterol metabolism and upregulated Cx43 expression and phosphorylation in tubules.

Distinctive actions of connexin 46 and connexin 50 in anterior pituitary folliculostellate cells.

Folliculostellate cell gap junctions establish a network for the transmission of information within the anterior pituitary. Connexins make up gap junction channels. Changes in connexin (Cx) turnover modify gap junction-mediated intercellular communication. We have reported that cytokines and hormones influence Cx43 turnover and coupling in folliculostellate cells and in the folliculostellate cell line TtT/GF. In addition, the expression of different connexins alters intercellular communication and connexins may have functions besides cell coupling. Here we assessed the expression, turnover and subcellular localization of Cx46 and Cx50 in the anterior pituitary and TtT/GF cells. Then, we assessed the impact of various natural (lactation, annual reproductive cycle, bFGF) and pathological (autoimmune orchitis, diabetes/obesity) conditions associated with altered anterior pituitary hormone secretion on Cx46 and Cx50. Anterior pituitary Cx46 and Cx50 expression and subcellular distribution were cell-dependent. Cx46 was expressed by folliculostellate, TtT/GF and endocrine cells. In the cytoplasm, Cx46 was chiefly associated with lysosomes. Variously sized Cx46 molecules were recovered exclusively in the TtT/GF cell nuclear fraction. In the nucleus, Cx46 co-localized with Nopp-140, a nucleolar factor involved in rRNA processing. Neither cytoplasmic nor nuclear Cx46 and Cx43 co-localized. Cx50 localized to folliculostellate and TtT/GF cells, and to the walls of blood capillaries, not to endocrine cells. Cx50 was cytoplasmic and associated with the cell membrane, not nuclear. Cx50 did not co-localize with Cx46 but it co-localized in the cytoplasm and co-immunoprecipitated with Cx43. Cx46 and Cx50 responses to various physiological and pathological challenges were different, often opposite. Cx46 and Cx43 expression and phosphorylation profiles differed in the anterior pituitary, whereas Cx50 and Cx43 were similar. The data suggest that Cx46 participates to cellular growth and proliferation and that Cx50, together with Cx43, contributes to folliculostellate cell coupling.

Myosin IIB deficiency in embryonic fibroblasts affects regulators and core members of the par polarity complex.

Wild-type (WT) and myosin heavy chain IIB null [MHCIIB (-/-)] embryonic fibroblasts were used as an experimental model to assess the role of the isoform B of myosin II (MII) in the regulation of the cell shape and intrinsic polarity. Genetic ablation of MHCIIB causes a persistent albeit, unstable protrusive activity in embryonic fibroblasts (Lo et al. in Nonmuscle myosin IIB is involved in the guidance of fibroblast migration. Mol Biol Cell 15:982-989, 2004). Here, we show that MHCIIB-deficient fibroblasts are characterized by a sustained guanine nucleotide exchange factor (GEF)-dependent activation of the small GTPase Rac-1 that is responsible for the continual lamellipodium formation. Moreover, we observed a sustained PKC-ζ activation and an increased association of cortactin with the plasma membrane in the MHCIIB (-/-) cells that were also dependent on GEF-mediated Rac-1 activation. Rac-1 activation and its downstream effects were induced in WT fibroblasts by inhibiting MII ATPase and crosslinking activities, suggesting that an altered actin-MII interaction favours Rac-1 activation, regardless of the MII isoform implicated. In addition, we found MIIB isoform-specific effects that were independent of Rac-1 activation. MHCIIA interacts with cortactin whereas MHCIIB does not. By contrast, MHCIIB interacts with Lgl1, a member of the Scribble/Dlg/Lgl polarity complex, whereas MHCIIA does not. MHCIIB (-/-) fibroblasts exhibited deregulated endogenous levels of the Par polarity complex members, Par3 and Par6. Together, the data show that MHCIIB deficiency causes imbalances in signalling pathways that are responsible for cell polarity determination. The results suggest that these pathways are targets of MIIB in the regulation of the cell's shape and polarity.

Cortactin/tyrosine-phosphorylated cortactin interaction with connexin 43 in mouse seminiferous tubules.

Deletion of the cortactin gene leads to male infertility. Considering that cortactin is an actin filament (F-actin)-binding protein associated with intercellular junctions, we measured changes in the expression and distribution of cortactin and tyrosine phosphorylated cortactin (P-cortactin) in the seminiferous epithelium of developing and adult mice to address the physiological significance of cortactin to germ cell differentiation. Cortactin was expressed in neonatal and developing Sertoli cells. Cortactin levels decreased early during puberty, while P-cortactin increased. Cortactin labeling was intense in the basal and apical thirds of the epithelium. Sertoli cell cytoplasmic processes facing spermatogonia, preleptotene spermatocytes, and step 8-13 spermatids were intensely labeled by both cortactin and P-cortactin. In contrast, the middle region of Sertoli cells exhibited diffuse cortactin labeling but no P-cortactin. This is consistent with the view that plasma membrane segments facing germ cells are part of the continuum of Sertoli cell junctional complexes that extend over lateral and apical membranes of supporting cells. Moreover, F-actin and P-cortactin share a common location in the seminiferous epithelium. The increased P-cortactin levels detected during puberty may be related to the modulatory effect of cortactin tyrosine phosphorylation on actin assembly at sites of selected Sertoli cell-germ cell contacts. Cortactin and connexin 43 (Cx43) were physically linked in seminiferous tubule homogenates and their colocalization in the basal and apical thirds of the seminiferous epithelium was stage-dependent. Our results suggest that cortactin-Cx43 interaction helps coordinate formation of cell-to-cell junctions and organization of the subsurface actin cytoskeleton in specific regions of the epithelium.

Defects in the regulatory clearance mechanisms favor the breakdown of self-tolerance during spontaneous autoimmune orchitis.

We identified aberrations leading to spontaneous autoimmune orchitis (AIO) in mink, a seasonal breeder and natural model for autoimmunity. This study provides evidence favoring the view that a malfunction of the clearance mechanisms for apoptotic cell debris arising from imbalances in phagocyte receptors or cytokines acting on Sertoli cells constitutes a major factor leading to breakdown of self-tolerance during spontaneous AIO. Serum anti-sperm antibody titers measured by ELISA reflected spermatogenic activity without causing immune inflammatory responses. Orchitic mink showed excess antibody production accompanied by spermatogenic arrest, testicular leukocyte infiltration, and infertility. AIO serum labeled the postacrosomal region, the mid and end piece of mink sperm, whereas normal mink serum did not. Normal serum labeled plasma membranes, whereas AIO serum reacted with germ cell nuclei. Western blot analyses revealed that AIO serum reacted specifically to a 23- and 50-kDa protein. The number of apostain-labeled apoptotic cells was significantly higher in orchitic compared with normal tubules. However, apoptosis levels measured by ELISA in seminiferous tubular fractions (STf) were not significantly different in normal and orchitic tubules. The levels of CD36, TNF-alpha, TNF-alpha RI, IL-6, and Fas but not Fas-ligand (L), and ATP-binding cassette transporter ABCA1 were changed in AIO STf. TNF-alpha and IL-6 serum levels were increased during AIO. Fas localized to germ cells, Sertoli cells, and the lamina propria of the tubules and Fas-L, to germ cells. Fas colocalized with Fas-L in residual bodies in normal testis and in giant cells and infiltrating leukocytes in orchitic tubules.

Isoform B of myosin II heavy chain mediates actomyosin contractility during TNFalpha-induced apoptosis.

Cells that are treated long-term with TNFalpha or short-term with TGFalpha together with cycloheximide (CHX) undergo apoptosis. Cell shrinkage and detachment during apoptosis is dependent on actomyosin contractility. Myosin II heavy chain (MHCII) isoforms have shared and distinct functions. Here, we investigated whether the involvement of MHCII isoforms A and B (MHCIIA and MHCIIB, respectively) in cell shrinkage and detachment differs during apoptosis. We show that TNFalpha induces caspase-dependent MHCIIA degradation, whereas MHCIIB levels and association with the cytoskeleton remained virtually unchanged in TtT/GF cells and NIH 3T3 fibroblasts. MHCIIA proteolysis also occurred in fibroblasts that lack MHCIIB when treated with TNFalpha and CHX together. The absence of MHCIIB did not affect cell death rate. However, MHCIIB-/- cells showed more resistance to TNFalpha-induced actin disassembly, cell shrinkage and detachment than wild-type fibroblasts, indicating the participation of MHCIIB in these events. Moreover, inhibition of atypical PKCzeta, which targets MHCIIB but not MHCIIA, blocked TNFalpha-induced shrinkage and detachment in TtT/GF cells and wild-type fibroblasts, but the inhibitory effect was significantly reduced in MHCIIB-/- fibroblasts. TNFalpha treatment increased cytoskeleton-associated myosin light chain (MLC) phosphorylation but did not induce actin cleavage. In conclusion, our results demonstrate that MHCIIB, together with MLC phosphorylation and actin, constitute the actomyosin cytoskeleton that mediates contractility during apoptosis.

Tumor necrosis factor-alpha-induced anterior pituitary folliculostellate TtT/GF cell uncoupling is mediated by connexin 43 dephosphorylation.

The anterior pituitary folliculostellate (FS) cells are key elements of the paracrine control of the pituitary function. These cells are the source and the target of growth factors and cytokines, and are connected to other pituitary cells via Cx43-mediated gap junctions. Here, we show that acute treatment of the FS TtT/GF cell line with TNF-alpha caused a transient cell uncoupling that was accompanied by the dephosphorylation of Cx43 in Ser368. These TNF-alpha-evoked effects were dependent on protein phosphatase 2A (PP2A) and protein kinase C (PKC) activities. TNF-alpha did not affect total cell Cx43-PP2A catalytic subunit interaction, but it did induce PP2A catalytic subunit recruitment to the Triton X-100 insoluble subcellular fraction, in which Cx43-gap junction plaques are recovered. This recruitment temporally coincided with Cx43 phosphorylated in Ser368-Cx43 dephosphorylation. Cx43 did not interact with the conventional PKC-alpha, but it did interact with the atypical PKC-zeta. Moreover, this interaction was weakened by TNF-alpha. Cx43 dephosphorylation in Ser368 was followed by the tyrosine phosphorylation of the protein. The temporary closure of gap junctions during acute TNF-alpha challenge may constitute a protective mechanism to limit or confine the spread of inflammatory signals among the FS cells.

The predominance of one of the SR-BI isoforms is associated with increased esterified cholesterol levels not apoptosis in mink testis.

Scavenger receptor class B type I (SR-BI) contributes to HDL-mediated cellular cholesterol efflux and is a phagocytosis-inducing phospholipid phosphatidylserine receptor in rat Sertoli cells, whereas the spliced variant of the SR-B gene, SR-BII, is implicated in the efflux of free cholesterol in macrophages. This study aimed to assess whether spontaneous autoimmune orchitis (AIO), which causes impaired clearance of apoptotic germ cells and spermatogenic arrest, involves SR-BI, SR-BII, and/or cholesterol. The levels measured during development and the annual reproductive cycle in normal mink were compared with those in mink with spontaneous AIO. Time periods with lowest tubular esterified cholesterol (EC) levels showed maximal SR-BI and SR-BII levels, and the periods when one or the other SR-BI isoform predominated showed increased EC levels and spermatogenic arrest in normal mink seminiferous tubules. In tubules with AIO, the predominance of only one or the other SR-BI isoform was the reverse of that measured in normal tubules, and it was associated with an increase in EC levels but not with apoptosis levels. SR-BI and SR-BII levels were not correlated with serum testosterone levels. SR-BI mainly localized to the Leydig cell, germ cell, and Sertoli cell surface, where its distribution was stage-specific. SR-BII was principally intracellular. Tubules from testes with AIO showed a deregulation of cholesterol homeostasis and SR-BI expression but relatively unchanged apoptosis levels. These results suggest that the expression of both SR-BI isoforms is required for the maintenance of low EC levels and that the predominance of only one isoform is associated with the accumulation of EC but not with apoptosis in the tubules.

Modulation of GJA1 turnover and intercellular communication by proinflammatory cytokines in the anterior pituitary folliculostellate cell line TtT/GF.

Our previous studies have advanced the idea that the folliculostellate cell GJA1 (gap junction membrane channel protein alpha1; previously known as connexin 43)-mediated gap junctions contribute to the establishment of an intercellular network that regulates the paracrine messages and the endocrine response within the anterior pituitary. The folliculostellate cells are targets for growth factors and cytokines that modulate hormone secretion. Proinflammatory cytokines modulate the cell-to-cell communication in many tissues of the body. The present study measured the effect of the proinflammatory cytokines tumor necrosis factor and interleukin-1 on the GJA1-mediated intercellular communication, specifically the expression, localization, degradation, and phosphorylation status of GJA1 in the folliculostellate cell line TtT/GF. The GJA1 localized to the plasma membrane and to minute cytoplasmic vesicles in the perinuclear area. Using different antibodies that recognize distinctly the nonphosphorylated from the phosphorylated forms of GJA1, we showed that nonphosphorylated GJA1 in Ser-368 (NP-GJA1) localized chiefly in the cytoplasm, whereas GJA1 phosphorylated in Ser-368 (P-GJA1) localized to the plasma membrane in controls. The cytokine treatment transiently increased 1) GJA1, NP-GJA1, and P-GJA1 levels; 2) NP-GJA1 and P-GJA1 degradation by both the lysosomal and proteasomal pathways; and 3) cell-to-cell communication in TtT/GF cells. The results suggest that the cytokine-evoked, transient enhancement of folliculostellate cell-mediated intercellular communication contributes to the coordination of the response among folliculostellate cells.

Actin cytoskeleton remodelling in the anterior pituitary folliculostellate cell line TtT/GF: participation of the actin-binding protein cortactin.

We have previously shown that the folliculostellate (FS) cells of the anterior pituitary change their shape from stellate (type I) to polygonal (type II) coincidently with variations in the secretory activity of the pituitary. To elucidate the mechanisms involved in this switch in phenotypes, here we studied the impact of serum factors on the morphology of the FS cell line TtT/GF. TtT/GF cells cultured in serum-containing medium displayed elongated shapes and membrane ruffles similarly to type I cells. Serum deprivation caused the loss of plasma membrane activity and the acquisition by the cells of a sedentary phenotype and of a polygonal shape typical of type II FS cells. Addition of serum to the starved cells induced the reappearance of membrane raffles and lamellipodia. The switch in phenotypes and the maintenance of a motile phenotype depended on tyrosine kinase but not on Erk activity. Because the transition between phenotypes involved the tyrosine kinase-dependent reorganization of cortical actin filaments, we studied the participation of the actin-binding protein, cortactin, a tyrosine kinase substrate. Cortactin and its tyrosine-phosphorylated form, pY421-cortactin, localized to membrane ruffles and lamellipodia in serum-cultured TtT/GF cells, while they were evenly distributed over the whole cell cortex in serum-starved cells. Serum treatment of starved cells induced a transient increase in pY421-cortactin levels and the clustering of pY421-cortactin in membrane regions where protrusions were developing. Both serum responses were blocked by a tyrosine kinase inhibitor. Together, the results indicate that the transition from a polygonal to an elongated shape entails the acquisition of a dynamic cortical actin cytoskeleton that involves the tyrosine kinase-dependent phosphorylation of cortactin and the translocation of cortical pY421-cortactin to sites of ruffle formation at the plasma membrane.

Relationship of the hormone-sensitive lipase-mediated modulation of cholesterol metabolism in individual compartments of the testis to serum pituitary hormone and testosterone concentrations in a seasonal breeder, the mink (Mustela vison).

The role of cholesterol differs in the two compartments of the testis. In the interstitial tissue, cholesterol is necessary for the synthesis of testosterone, whereas in the seminiferous tubules, membrane cholesterol content in developing germ cells will influence the gametes' fertility. Here we evaluate the hormone-sensitive lipase (HSL) modulation of the cholesterol metabolism in each compartment of the testis. Two HSL immunoreactive bands of 104- and 108-kDa were detected in Western blots performed with polyclonal anti-human HSL antibodies in the interstitial tissue (ITf)- and seminiferous tubule (STf)-enriched fractions generated from testes harvested at 30-day intervals during puberty and, in the adult mink, during the annual seasonal reproductive cycle. Epididymal spermatozoa expressed a 104-kDa HSL isoform, and HSL was active in these cells. Immunolabeling localized HSL to interstitial macrophages; Sertoli cells, where its distribution was stage specific; spermatids; and the equatorial segment of spermatozoa. Total HSL protein levels, specific enzymatic activity, and free cholesterol (FC):esterified cholesterol (EC) ratios varied concomitantly in STf and ITf and reached maximal values in the adult during the period of maximal spermatogenic activity. In STf, HSL-specific activity correlated with FC:EC ratios but not with triglyceride levels. In STf, high HSL-specific activity occurred concomitantly with high FSH serum levels. In ITf, HSL-specific activity was high during periods of low serum prolactin levels and high serum testosterone levels. The results suggest that 1) modulation of cholesterol metabolism in individual testicular compartments may be regulated by HSL isoforms expressed by distinct cells; 2) interstitial macrophages may be part of a system involved in the synthesis of steroid hormones and in the recycling of sterols in the interstitium, whereas in the tubules, recycling could be ensured by Sertoli cells; 3) there is distinctive substrate preference for testicular HSL; and 4) HSL may be the only cholesterol esterase in this location.