Connexin 43 is critical to maintain the homeostasis of the blood-testis barrier via its effects on tight junction reassembly

In mammalian testes, the blood-testis barrier (BTB) or Sertoli cell barrier created by specialized junctions between Sertoli cells near the basement membrane confers an immunological barrier by sequestering the events of meiotic division and postmeiotic germ cell development from the systemic circulation. The BTB is constituted by coexisting tight junctions (TJs), basal ectoplasmic specializations, desmosomes, and gap junctions. Despite being one of the tightest blood-tissue barriers, the BTB has to restructure cyclically during spermatogenesis. A recent study showed that gap junction protein connexin 43 (Cx43) and desmosome protein plakophilin-2 are working synergistically to modulate the BTB integrity by regulating the distribution of TJ-associated proteins at the Sertoli-Sertoli cell interface. However, the precise role of Cx43 in regulating the cyclical restructuring of junctions remains obscure. In this report, the calcium switch and the bisphenol A (BPA) models were used to induce junction restructuring in primary cultures of Sertoli cells isolated from rat testes that formed a TJ-permeability barrier that mimicked the BTB in vivo. The removal of calcium by EGTA perturbed the Sertoli cell tight junction barrier, but calcium repletion allowed the "resealing" of the disrupted barrier. However, a knockdown of Cx43 in Sertoli cells by RNAi significantly reduced the kinetics of TJ-barrier resealing. These observations were confirmed using the bisphenol A model in which the knockdown of Cx43 by RNAi also perturbed the TJ-barrier reassembly following BPA removal. In summary, Cx43 is crucial for TJ reassembly at the BTB during its cyclic restructuring throughout the seminiferous epithelial cycle of spermatogenesis.     

Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study

Nitric oxide (NO) synthase (NOS) catalyzes the oxidation of L-arginine to NO. NO plays a crucial role in regulating various physiological functions, possibly including junction dynamics via its effects on cAMP and cGMP, which are known modulators of tight junction (TJ) dynamics. Although inducible NOS (iNOS) and endothelial NOS (eNOS) are found in the testis and have been implicated in the regulation of spermatogenesis, their role(s) in TJ dynamics, if any, is not known. When Sertoli cells were cultured at 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units, functional TJ barrier was formed when the barrier function was assessed by quantifying transepithelial electrical resistance across the cell epithelium. The assembly of the TJ barrier was shown to associate with a significant plummeting in the levels of iNOS and eNOS, seemingly suggesting that their presence by producing NO might perturb TJ assembly. To further confirm the role of NOS on the TJ barrier function in vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitor and a soluble guanylate cyclase inhibitor, was added to the Sertoli cell cultures during TJ assembly. Indeed, ZnPP was found to facilitate the assembly and maintenance of the Sertoli cell TJ barrier, possibly by inducing the production of TJ-associated proteins, such as occludin. Subsequent studies by immunoprecipitation and immunoblotting have shown that iNOS and eNOS are structurally linked to TJ-integral membrane proteins, such as occludin, and cytoskeletal proteins, such as actin, vimentin, and alpha-tubulin. When the cAMP and cGMP levels in these ZnPP-treated samples were quantified, a ZnPP-induced reduction of intracellular cGMP, but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP, a cell membrane-permeable analog of cGMP, could also perturb the TJ barrier dose dependently similar to the effects of 8-bromo-cAMP. KT-5823, a specific inhibitor of protein kinase G, was shown to facilitate the Sertoli cell TJ barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.     

Is cadmium chloride-induced inter-sertoli tight junction permeability barrier disruption a suitable in vitro model to study the events of junction disassembly during spermatogenesis in the rat testis?

The events of germ cell movement during spermatogenesis are composed of intermittent phases of junction disassembly and reassembly. Although primary Sertoli cells cultured in vitro can be used to study junction reassembly, an in vitro model to study the events of junction disassembly is still lacking. We have assessed whether the CdCl(2)-induced inter-Sertoli tight junction (TJ) permeability barrier disruption in vitro can fill this gap. When Sertoli cells (1.2 x 10(6) cells/cm(2)) were cultured on Matrigel-coated bicameral units to allow the assembly of inter-Sertoli TJs, it was manifested by a steady rise in transepithelial electrical resistance across the Sertoli cell epithelia. Exposure of these cells on day 1 (i.e. 24 h after their isolation) to CdCl(2) at 5-10 microM for 8 h could perturb the inter-Sertoli TJ assembly dose dependently without any apparent cytotoxicity. Likewise, when cells were exposed to CdCl(2) (0.1-5 microM) on day 4 for 8 h after inter-Sertoli TJs were already assembled, CdCl(2) also perturbed the maintenance of inter-Sertoli TJ permeability barrier dose dependently without signs of cell cytotoxicity. Although the perturbed inter-Sertoli TJs were not capable of resealing even after the removal of CdCl(2), the presence of testosterone (T) at 1 x 10(-9) M allowed resealing of the inter-Sertoli TJ barrier after CdCl(2) was removed, whereas the presence of 2 x 10(-7) M testosterone even protected Sertoli cells from CdCl(2)-induced damage. More important, the reassembly of inter-Sertoli TJs after CdCl(2)-induced TJ disruption was accompanied by changes in cellular gene expression of occludin and urokinase plasminogen activator, which mimicked their patterns during inter- Sertoli TJ assembly in vitro without CdCl(2) treatment. Based on these results, it is apparent that CdCl(2)-induced inter-Sertoli TJ disassembly is a potential in vitro model to study the events of junction disassembly.     

Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11

Throughout spermatogenesis, inter-Sertoli tight junctions (TJs) that create the blood-testis barrier in the rat must be disassembled and reassembled to permit the timely passage of preleptotene spermatocytes from the basal to the adluminal compartment of the seminiferous epithelium. However, the mechanism(s) and the participating molecules that regulate this event are largely unknown. Although there is no in vitro model to study the event and regulation of inter-Sertoli TJ disassembly, primary cultures of Sertoli cells in vitro can be used to study junction assembly. In this study, we sought to investigate whether cytokines are involved in the inter-Sertoli TJ assembly in vitro. Sertoli cells isolated from 20-day-old rats were cultured at a density of 0.5-1.2 x 10(6) cells/cm(2) on Matrigel-coated dishes or bicameral units for 8-9 days. The steady-state messenger RNA levels of basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-beta2, and TGF-beta3 at different time points were assessed by semiquantitative RT-PCR. In selected experiments, the assembly of inter-Sertoli TJs was monitored by transepithelial electrical resistance measurement. It was found that there was no change in the expression of basic fibroblast growth factor throughout the entire culture period. However, there was a 2-fold reduction in the expression of TGF-beta2 and TGF-beta3 at the time inter-Sertoli TJs were being assembled. On days 5-8, after the inter-Sertoli TJs had been assembled, the Sertoli cell steady-state messenger RNA levels of TGF-beta2 and TGF-beta3 increased by as much as 3- and 6-fold, respectively, when compared with Sertoli cells on days 1-3 when TJs were being assembled. Also, it was found that recombinant TGF-beta3 added to Sertoli cells cultured in vitro at 1.2 x 10(6) cells/cm(2) on Matrigel-coated bicameral units perturbed the inter-Sertoli TJ permeability barrier dose-dependently. Moreover, the presence of TGF-beta3 also inhibited the transient and/or basal expression of several TJ-associated proteins, which include occludin, zonula occludens-1, and claudin-11 when inter-Sertoli TJs were being assembled in vitro. These results suggest that TGF-beta plays a crucial role in regulating the complicated biochemical events of junction assembly in the testis.     

Suppression of tumor necrosis factor alpha-induced matrix metalloproteinase 9 production by the introduction of a super-repressor form of inhibitor of nuclear factor kappaBalpha complementary DNA into immortalized human salivary gland acinar cells. Prevention of the destruction of the acinar structure in Sjögren's syndrome salivary glands

OBJECTIVE: We have previously shown that specific enhancement in acinar cells of proteolytic activity induced by tumor necrosis factor alpha (TNFalpha) may be responsible for the destruction of the acinar structure in the salivary glands of patients with Sj?gren's syndrome. Because matrix metalloproteinase 9 (MMP-9) is regulated by nuclear factor kappaB (NF-kappaB), we investigated the effect of a super-repressor form of inhibitor of nuclear factor kappaBalpha (srIkappaBalpha) on the suppression of TNFalpha-induced MMP-9 production in acinar cells. METHODS: Two srIkappaBalpha complementary DNA (cDNA)-transfected acinar cell clones (ACMT-6 and ACMT-7) and 1 empty vector-transfected cell clone (ACpRc-1) were established. After treatment of cell clones with TNFalpha, the expression of MMP-9 was examined. In addition, the effect of TNFalpha on cell growth and the morphogenetic behavior of cell clones cultured on type IV collagen-coated dishes were examined. RESULTS: TNFalpha induced the production of MMP-9 in the ACpRc-1 cell clone, but greatly suppressed MMP-9 production in ACMT-6 and ACMT-7 clones. No apparent cytotoxic effect of TNFalpha treatment was observed in these cell clones. When ACpRc-1 cells were seeded on type IV collagen-coated dishes in the presence of both TNFalpha and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, even when ACMT-6 and ACMT-7 cells were cultured under the same culture conditions as those for ACpRc-1, these cell clones attached to the substrate and grew consistently without showing apoptosis. Conclusion. These observations indicate that suppression of TNFalpha-induced MMP-9 production by the introduction of srIkappaBalpha cDNA corrected the aberrant in vitro morphogenesis of acinar cells grown on type IV collagen.     

TGF-beta3 regulates the blood-testis barrier dynamics via the p38 mitogen activated protein (MAP) kinase pathway: an in vivo study

Recent studies using Sertoli cells cultured in vitro to permit tight junction (TJ) assembly have shown that TJ dynamics are regulated, at least in part, by TGF-beta3 via the p38 mitogen activated protein (MAP) kinase pathway. This in turn regulates the production of occludin, a TJ-integral membrane protein, by Sertoli cells. Yet it is not known if this pathways is used by Sertoli cells to regulate the blood-testis barrier (BTB) function in vivo. Using an in vivo model for studying BTB dynamics, we report herein the CdCl(2)-induced BTB damage in rats was associated with a significant reduction in testicular occludin along with a loss of immunoreactive occludin in the seminiferous epithelium at the site of the BTB. Also, this CdCl(2)-induced occludin loss from the BTB coincided with a surge in testicular TGF-beta3, as well as p-p38 MAP kinase (the phosphorylated/activated form of p38), but not p38 MAP kinase and neither extracellular signal-regulated kinase nor its phosphorylated form (ERK/p-ERK), consistent with results of in vitro studies. More important, intratesticular administration of SB202190, a specific p38 MAP kinase inhibitor, could block the CdCl(2)-induced occludin loss from the BTB. These results illustrate that BTB dynamics in vivo are regulated by the TGF-beta3/p38 MAP kinase pathway, which in turn determines the level of occludin at the site of Sertoli cells TJs.     

An autocrine axis in the testis that coordinates spermiation and blood-testis barrier restructuring during spermatogenesis

The mechanism(s) that regulate and coordinate the events of spermiation and blood-testis barrier (BTB) restructuring in the seminiferous epithelium that occur concurrently at stage VIII of the seminiferous epithelial cycle of spermatogenesis are unknown. In this report, fragments derived from the laminin complex composed of laminin alpha3, beta3, and gamma3 chains (laminin-333) at the apical ectoplasmic specialization (apical ES) were shown to modulate BTB dynamics directly and/or indirectly via hemidesmosome. Experiments were performed using cultured Sertoli cells with functional tight junction (TJ) barrier and the ultrastructural features of the BTB but not apical ES. Recombinant protein fragments of laminin beta3 and gamma3 chains were shown to reduce the protein levels of occludin and beta1-integrin dose dependently at the Sertoli-Sertoli and Sertoli-basement membrane interface, respectively, thereby destabilizing the BTB permeability function. These results were corroborated by transient overexpression of laminin fragments in Sertoli cells. To further assess the role of beta1-integrin in hemidesmosome, knockdown of beta1-integrin in Sertoli cells by RNAi was found to associate with occludin redistribution at the Sertoli-Sertoli cell interface, wherein occludin moved away from the cell surface and became associated with endosomes, thereby destabilizing the BTB. In short, an apical ES-BTB-hemidesmosome autocrine regulatory axis was identified in testes, coordinating the events of spermiation and BTB restructuring that occur at the opposite ends of the seminiferous epithelium during spermatogenesis.     

Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes

The timely restructuring of the blood-testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor alpha (TNFalpha), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFalpha directly to adult rat testes in vivo at 0.5 and 2 mug/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, alpha-, and beta-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFalpha treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFalpha by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring ('opening'?) of the BTB during spermatogenesis to facilitate germ cell migration.     

Suppression of tumor necrosis factor alpha-induced matrix metalloproteinase 9 production in human salivary gland acinar cells by cepharanthine occurs via down-regulation of nuclear factor kappaB: a possible therapeutic agent for preventing the destruction of the acinar structure in the salivary glands of Sjögren's syndrome patients

OBJECTIVE: Our previous results suggested that suppression of tumor necrosis factor alpha (TNFalpha)-induced matrix metalloproteinase 9 (MMP-9) could prevent the destruction of acinar tissue in the salivary glands of patients with Sj?gren's syndrome (SS). The present study was undertaken to investigate the effect of cepharanthine on the suppression of TNFalpha-induced MMP-9 production in NS-SV-AC, an SV40-immortalized normal human acinar cell clone. METHODS: After pretreatment with or without cepharanthine, NS-SV-AC cells were treated with TNFalpha alone or with a combination of TNFalpha and cepharanthine. The expression of MMP-9 was then examined at the protein and messenger RNA levels. In addition, the effect of cepharanthine on the morphogenetic behavior of NS-SV-AC cells cultured on type IV collagen-coated dishes in the presence of TNFalpha was examined. RESULTS: Although TNFalpha induced the production of MMP-9 in NS-SV-AC cells, this production was greatly suppressed when cells were pretreated with cepharanthine, followed by treatment with both TNFalpha and cepharanthine. In addition, cepharanthine suppressed the TNFalpha-stimulated NF-kappaB activity by partly preventing the degradation of IkappaBalpha protein in NS-SV-AC cells. When NS-SV-AC cells were seeded on type IV collagen-coated dishes in the presence of both TNFalpha and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, pretreatment with cepharanthine restored the aberrant in vitro morphogenesis of the NS-SV-AC cells. CONCLUSION: These results may indicate a molecular mechanism by which cepharanthine is able to protect against the destruction of the acinar structure in salivary glands from patients with SS.     

基质金属蛋白酶-9在心血管疾病中的研究进展

基质金属蛋白酶-9(MMP-9)又称明胶酶B,在病理情况下潜在型MMP-9被激活,参与许多心血管疾病的发生发展过程,如动脉粥样硬化斑块的形成、破裂,缺血再灌注损伤、心肌梗死后心室重构、充血性心力衰竭进展、心房颤动的发生及维持等。MMP-9/R279Q基因多态现象与稳定型心绞痛患者心血管事件的发生相关并且血浆MMP-9可以作为冠心病患者发生心血管病死亡的预测因子。     

Clinical significance of changes in tumor markers, extracellular matrix, MMP-9 and VEGF in patients with gastric carcinoma

BACKGROUND/AIMS: The aim of this study was to evaluate the prognostic significance of some serum tumor marker level, extracellular matrix (ECM), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) in patients with gastric cancer. METHODOLOGY: The serum tumor markers included CEA, CA50 and CA19-9, ECM included laminin (LN), hyaluronic acid (HA), and collagen type III and IV were measured in 40 patients with gastric carcinoma and 20 matched healthy controls by radioimmunoassay. MMP-9, VEGF and MVD were measured with immunohistochemical methods and the computer image analyzer. Microvascular density (MVD) in tissues of patients with gastric carcinoma was detected. RESULTS: The levels of CEA, CA50, CA19-9, HA, LN and collagen type IV in the patients with metastasis were significantly higher than those in the patients without metastasis (p < 0.05). The expression of MMP-9 and collage type IV in the patients with metastasis and poorly differentiated carcinomas were significantly higher than those in the patients without metastasis whose tumors were well/moderately differentiated (p < 0.05). CONCLUSIONS: CEA, CA50, CA19-9, HA, LN and collagen type IV levels can be used as a signal of metastasis and disease progression in patients with gastric carcinoma. When a gastric carcinoma expresses a high level of MMP-9 and VEGF with high MVD, the power of infiltration and metastasis of the gastric carcinoma is enhanced.     

Connexin 43 and plakophilin-2 as a protein complex that regulates blood–testis barrier dynamics

The blood–testis barrier (BTB) formed by adjacent Sertoli cells is composed of coexisting tight junction (TJ), basal ectoplasmic specialization (ES), and desmosome-like junction. Desmosome-like junctions display structural features of desmosome and gap junctions, but its function at the BTB remains unknown. Herein, we demonstrate that connexin 43 (Cx43), a gap junction integral membrane protein, structurally interacts with desmosomal protein plakophilin-2 (PKP2), basal ES proteins N-cadherin and β-catenin, and signaling molecule c-Src, but not with the TJ proteins occludin and ZO-1 in the seminiferous epithelium of adult rats. The localization of Cx43 in the seminiferous epithelium during (i) the normal epithelial cycle of spermatogenesis and (ii) anchoring junction restructuring at the Sertoli–spermatid interface induced by adjudin which mimics junction restructuring events during spermatogenesis have suggested that Cx43 is involved in cell adhesion. The knockdown of Cx43 by RNAi technique using specific siRNA duplexes was performed in primary Sertoli cell cultures with an established TJ permeability barrier that mimicked the BTB in vivo. This knockdown of Cx43 affected neither the TJ barrier function nor the steady-state levels of junction proteins of TJ, basal ES, and desmosome-like junction. However, after the knockdown of both Cx43 and PKP2, the Sertoli cell TJ barrier function was perturbed transiently. This perturbation was concomitant with a mislocalization of occludin and ZO-1 from the cell–cell interface. In summary, Cx43 and PKP2 form a protein complex within the desmosome-like junction to regulate cell adhesion at the BTB, partly through its effects on the occludin/ZO-1 complex, so as to facilitate the transit of primary preleptotene spermatocytes.     

Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9

ARH-77 human myeloma cells invade into type I collagen gels but become non-invasive when engineered to express syndecan-1, a heparan sulphate proteoglycan that promotes cell adhesion to collagen. To determine if syndecan-1 expression influences the activity of proteases that may facilitate invasion, we analysed media harvested from syndecan-1 expressing and non-expressing cells. High levels of a 92 kD gelatinase accumulated in serum-free growth medium of both parental and control-transfected ARH-77, but much less 92 kD gelatinase accumulated in the medium of ARH-77 transfectants expressing syndecan-1. The gelatinase was identified as matrix metalloproteinase (MMP)-9 because its activity was immunoprecipitated with a MMP-9-specific monoclonal antibody. Gelatinase activity and Western blot analyses revealed 2-3-fold less MMP-9 in medium from syndecan-1 transfected cells than in medium from parental cells. Decreased MMP-9 was not due to increased association of MMP-9 with cells expressing syndecan-1. An inverse correlation between the syndecan 1 level and the level of MMP-9 accumulation in the media was observed using a panel of ARH-77 transfectants expressing syndecan-1. Investigation of six unrelated human myeloma cell lines confirmed that high gelatinase levels were recovered from conditioned media of those that did not express syndecan-1 (ARH-77, Mer and Col) and one line that expressed a low level of syndecan-1 (RPMI-8226), but low gelatinase levels were recovered from media of lines that expressed high levels of syndecan-1 (ARK and clone 2+). Therefore syndecan-1 may play a dual role in inhibiting the metastasis of tumour cells by promoting cell adhesion to the extracellular matrix and suppressing the proteolytic activity needed for invasion.     

肝素对大鼠肝星状细胞生长、细胞外基质和基质金属蛋白酶基因表达的影响

目的 探讨肝素对ＨＳＣ生长、细胞外基质和基质金属蛋白酶基因表达的影响。方法 用肝素和不用肝素对活化的ＨＳＣ进行处理,以直接细胞计数和ＢｒｄＵ标记免疫细胞化学染色检测细胞的生长情况。用免疫细胞化学染色和细胞闰核酸分子杂交分别检测Ｉ、Ⅳ型前胶原蛋白、纤连蛋白和Ⅰ、Ⅳ型胶原、纤连蛋白、基质金属蛋白酶－２及膜型基质金属蛋白酶基因的表达,并用酶图法检测基质金属蛋白酶－２的活性。结果 肝素使血清所致的ＨＳＣ生     

Evidence that MMP-2 and TIMP-2 are at play in the FSH-induced changes in Sertoli cells

FSH has been shown to elicit in vitro changes in the Sertoli cell cytoskeleton through involving proteases, and cytochalasin-D mimics FSH. Testis extracts were screened (RT-PCR) for various metalloproteinases (MMPs), 20-day-old rat Sertoli cells were purified, cultured and treated with FSH, cytochalasin D and TNFalpha (to antagonize FSH action). Cell shape (phase-contrast microscopy) and levels for MMP-2 (gelatin zymography) and its inhibitor TIMP-2 (Northern and Western blot) were monitored. TNFalpha-treated cells spread readily and grew larger than FSH-treated cells. Cytochalasin-D mimicked FSH, and MMP-2 production and TIMP-2 gene expression were augmented. Interestingly, TNFalpha reversed FSH- and cytochalasin D-induced effects both on cell shape and on MMP-2 and TIMP-2. These effects occurred during the first 48 h of culture, when Sertoli cells migrated from the freshly dispersed aggregates, but not once cells were organized in monolayers. MMP-2 and TIMP-2 are likely involved in the FSH-induced changes in Sertoli cells.     

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

In the testis, the blood–testis barrier (BTB) is constituted by specialized junctions between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. Although the BTB is one of the tightest blood–tissue barriers in the mammalian body, it undergoes extensive restructuring at stage VIII of the seminiferous epithelial cycle to facilitate the transit of preleptotene spermatocytes. Thus, meiosis and postmeiotic germ cell development take place in the seminiferous epithelium behind the BTB. Cytokines (e.g., TGF-β3) are known to regulate BTB dynamics by enhancing the endocytosis of integral membrane proteins and their intracellular degradation. This thus reduces the levels of proteins above the spermatocytes in transit at the BTB, causing its disruption after testosterone-induced new tight junction (TJ) fibrils are formed behind these cells. By using Sertoli cells cultured in vitro with an established TJ permeability barrier that mimicked the BTB in vivo, Cdc42 was shown to be a crucial regulator that mediated the TGF-β3–induced BTB disruption. TGF-β3 was shown to activate Cdc42 to its active GTP-bound form. However, an inactivation of Cdc42 by overexpressing its dominant-negative mutant T17N in Sertoli cell epithelium was shown to block the TGF-β3–induced acceleration in protein endocytosis. Consequently, this prevented the disruption of Sertoli cell TJ permeability barrier and redistribution of TJ proteins (e.g., CAR and ZO-1) from the cell–cell interface to cell cytosol caused by TGF-β3. In summary, Cdc42 is a crucial regulatory component in the TGF-β3–mediated cascade of events that leads to the disruption of the TJ fibrils above the preleptotene spermatocytes to facilitate their transit.     

Combination of tumor necrosis factor-alpha ablation and matrix metalloproteinase inhibition prevents heart failure after pressure overload in tissue inhibitor of metalloproteinase-3 knock-out mice

Cytokine and extracellular matrix (ECM) homeostasis are distinct systems that are each dysregulated in heart failure. Here we show that tissue inhibitor of metalloproteinase (TIMP)-3 is a critical regulator of both systems in a mouse model of left ventricular (LV) dilation and dysfunction. Timp-3(-/-) mice develop precipitous LV dilation and dysfunction reminiscent of dilated cardiomyopathy (DCM), culminating in early onset of heart failure by 6 weeks, compared with wild-type aortic-banding (AB). Timp-3 deficiency resulted in increased TNFalpha converting enzyme (TACE) activity within 6 hours after AB leading to enhanced tumor necrosis factor-alpha (TNFalpha) processing. In addition, TNFalpha production increased in timp-3(-/-)-AB myocardium. A significant elevation in gelatinase and collagenase activities was observed 1 week after AB, with localized ECM degradation in timp-3(-/-)-AB myocardium. Timp-3(-/-)/tnfalpha(-/-) mice were generated and subjected to AB for comparative analyses with timp-3(-/-)-AB mice. This revealed the critical role of TNFalpha in the early phase of LV remodeling, de novo expression of Matrix metalloproteinases (MMP)-8 in the absence of TNFalpha, and highlighted the importance of interstitial collagenases (MMP-2, MMP-13, and MT1-MMP) for cardiac ECM degradation. Ablation of TNFalpha, or limiting MMP activity with a synthetic MMP inhibitor (PD166793), each partially attenuated LV dilation and cardiac dysfunction in timp-3(-/-)-AB mice. Notably, combining TNFalpha ablation with MMP inhibition completely rescued heart disease in timp-3(-/-)-AB mice. This study provides a basis for anti-TNFalpha and MMP inhibitor combination therapy in heart disease.