Randall H. Renegar

Inflammation and Disruption of the Mucosal Architecture in Claudin-7–Deficient Mice

BACKGROUND & AIMS Integrity of the intestinal epithelium is required for nutrition absorption and defense against pathogens. Claudins are cell adhesion molecules that localize at tight junctions (TJs); many are expressed in the intestinal tract, but little is known about their functions. Claudin-7 is unique in that it has a stronger basolateral membrane distribution than other claudins, which localize primarily to apical TJs in the intestinal epithelium. We investigated the basolateral functions of claudin-7 and assessed the effects of disruption of Cldn7 in intestines of mice. METHODS We generated Cldn7(-/-) mice and examined their intestines by histology, molecular and cellular biology, and biochemistry approaches. We performed gene silencing experiments in epithelial cell lines using small interfering RNAs (siRNAs). RESULTS The Cldn7(-/-) mice had severe intestinal defects that included mucosal ulcerations, epithelial cell sloughing, and inflammation. Intestines of Cldn7(-/-) mice produced significantly higher levels of cytokines, the nuclear factor κB p65 subunit, and cyclooxygenase 2; they also up-regulated expression of matrix metalloproteinases (MMPs)-3 and -7. siRNA in epithelial cell lines showed that the increased expression of MMP-3 resulted directly from claudin-7 depletion, whereas that of MMP-7 resulted from inflammation. Electron microscopy analysis showed that intestines of Cldn7(-/-) mice had intercellular gaps below TJs and cell matrix loosening. Deletion of Cldn7 reduced expression and altered localization of the integrin α2 subunit in addition to disrupting formation of complexes of claudin-7, integrin α2, and claudin-1 that normally form in epithelial basolateral compartments of intestines. CONCLUSIONS In mice, claudin-7 has non-TJ functions, including maintenance of epithelial cell-matrix interactions and intestinal homeostasis.

Retinoic Acid Induces Multiple Hallmarks of the Prospermatogonia-to-Spermatogonia Transition in the Neonatal Mouse

ABSTRACT In mammals, most neonatal male germ cells (prospermatogonia) are quiescent and located in the center of the testis cords. In response to an unknown signal, prospermatogonia transition into spermatogonia, reenter the cell cycle, divide, and move to the periphery of the testis cords. In mice, these events occur by 3–4 days postpartum (dpp), which temporally coincides with the onset of retinoic acid (RA) signaling in the neonatal testis. RA has a pivotal role in initiating germ cell entry into meiosis in both sexes, yet little is known about the mechanisms and about cellular changes downstream of RA signaling. We examined the role of RA in mediating the prospermatogonia-to-spermatogonia transition in vivo and found 24 h of precocious RA exposure-induced germ cell changes mimicking those that occur during the endogenous transition at 3–4 dpp. These changes included: 1) spermatogonia proliferation; 2) maturation of cellular organelles; and 3), expression of markers characteristic of differentiating spermatogonia. We found that germ cell exposure to RA did not lead to cellular loss from apoptosis but rather resulted in a delay of ∼2 days in their entry into meiosis. Taken together, our results indicate that exogenous RA induces multiple hallmarks of the transition of prospermatogonia to spermatogonia prior to their entry into meiosis.

Hydroxycamptothecin-loaded Fe3O4 nanoparticles induce human lung cancer cell apoptosis through caspase-8 pathway activation and disrupt tight junctions.

10‐Hydroxycamptothecin (HCPT) elicits strong anti‐cancer effects and is less toxic than camptothecin (CPT), making it widely used in recent clinical trials. However, its low solubility limits its application as an effective anti‐cancer therapy. In the present study we investigate the hypothesis that the unique water dispersible oleic acid‐Triton X‐100‐coated Fe3O4 nanoparticles loaded with HCPT disrupt epithelial cell–cell junctions and induce human lung cancer cell apoptosis through the caspase‐8 pathway. We characterized the HCPT‐loaded nanoparticles and determined their effects on lung cancer cell viability and apoptosis by using immunofluorescence light microscopy and SDS‐PAGE/immunoblots. We found that HCPT‐loaded nanoparticles elicited an anti‐proliferative effect in a dose‐dependent manner. HCPT‐loaded nanoparticles reduced the expression of cell–cell junction protein claudins, E‐cadherin and ZO‐1, and transmission electron microcopy demonstrated a disrupted tight junction ultrastructure. Transepithelial electric resistance was also reduced, indicating the reduction of tight junction functions. The HCPT‐loaded nanoparticles increased phosphorylation of p38 and SAPK/JNK while it showed no effects on p42/44 MAP kinase. Compared with void Fe3O4 nanoparticles or HCPT drug alone, HCPT drug‐loaded nanoparticles evoked synergistic effects by increasing cell apoptosis with enhanced activation of the caspase‐8 pathway. Therefore, our current study highlights the potential of HCPT drug‐loaded nanoparticles as a chemotherapeutic agent for increasing anti‐cancer drug efficacy. (Cancer Sci 2011; 102: 1216–1222)

Effects of peripheral nerve lesions during pregnancy on parturition in rats

SummaryBilateral section of either the sensory or motor branch of the pelvic nerve or pudendal nerve was performed in rats on days 8–10 of pregnancy, and the effects on delivery were observed. Bilateral resection of the sensory branch of the pelvic nerve reduced the number of live pups per litter, and increased the number of stillbirths and the number of fetuses retained in utero per litter at day 24. Sectioning motor components of the pelvic nerve, or both motor and sensory components of the pudendal nerve, had no effects on delivery in rats. We conclude that of the peripheral nerves evaluated in this study, only the sensory branch of the pelvic nerve is required for normal vaginal delivery in this species.

A non-tight junction function of claudin- 7—Interaction with integrin signaling in suppressing lung cancer cell proliferation and detachment

BackgroundClaudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells.MethodsClaudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β1. Tumor growth in vivo were analyzed using athymic nude mice.ResultsClaudin-7 co-localizes and forms a stable complex with integrin β1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β1 expression and increases cell-matrix adhesion. Integrin β1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells.ConclusionIn this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β1.

Expression and localization of prohormone convertase 1/3 (SPC3) in porcine ovary

Tissue distribution and cellular localization of PC1/3 mRNA in porcine tissues were examined by ribonuclease protection assay and in situ hybridization. PC1/3 mRNA was detected mainly in the corpus luteum of pregnant sow and brain. Within the ovary, PC1/3 and relaxin transcripts colocalized within large luteal cells. Levels of PC1/3 transcripts in corpora lutea increased as gestation advanced, parallel with an observed increase in relaxin transcripts. A role for PC1/3 in proprotein processing in the ovary is discussed. Mol. Reprod. Dev. 57:361–365, 2000.

Nuclear localization of the actin regulatory protein Palladin in sertoli cells.

In the testis, F‐actin structures are involved in spermatid nuclear remodeling and cytoplasm reduction, maintenance of the blood–testis barrier, support of the spermatogonial stem cell niche, and release of spermatids into the tubular lumen. To gain a better understanding of actin regulation in Sertoli–germ cell interactions, we investigated the expression of the Palladin (Palld) gene, which encodes a widely expressed phosphoprotein that localizes to actin‐rich cytoplasmic structures, including focal adhesions, cell–cell junctions, podosomes, and stress fibers, and serves as a molecular scaffold to bundle actin fibers. In germ cells, PALLD was concentrated along the tubulin‐ and F‐actin‐containing cytoplasmic manchette that forms adjacent to the elongating spermatid nucleus during spermiogenesis. To our surprise, PALLD relocated from the cytoplasm to the nucleus of Sertoli cells in the juvenile testis, coincident with the onset of puberty, and this localization was maintained in the adult. We provide evidence that the 140 kDa isoform of PALLD predominates in Sertoli cells, and that it is apparently cleaved, with the C‐terminus localizing to the nucleus while the N‐terminus remains cytoplasmic. We investigated the nuclear localization of the C‐terminus of PALLD and found that it is regulated by a putative nuclear export signal. These results provide the foundation for future work employing Sertoli cell‐ and spermatid‐specific Palld‐knockout mice to study diverse roles of PALLD as both a nuclear‐actin regulatory protein and as a potential regulator of manchette formation during spermatogenesis. Mol. Reprod. Dev. 80: 403–413, 2013.

Localization of the actin-binding protein fesselin in chicken smooth muscle

This report compares cellular localization of fesselin in chicken smooth, skeletal and cardiac muscle tissues using affinity purified polyclonal fesselin antibodies. Western blot analyses revealed large amounts of fesselin in gizzard smooth muscle with lower amounts in skeletal and cardiac muscle. In gizzard, fesselin was detected by immunofluorescence as discrete cytoplasmic structures. Fesselin did not co-localize with talin, vinculin or caveolin indicating that fesselin is not associated with dense plaques or caveolar regions of the cell membrane. Immunoelectron microscopy established localization of fesselin within dense bodies. Since dense bodies function as anchorage points for actin and desmin in smooth muscle cells, fesselin may be involved in establishing cytoskeletal structure in this tissue. In skeletal muscle, fesselin was associated with desmin in regularly spaced bands distributed along the length of muscle fibers suggesting localization to the Z-line. Infrequently, this banding pattern was observed in heart tissue as well. Localization at the Z-line of skeletal and cardiac muscle suggests a role in contraction of these tissues.

Measurement of Plasma and Tissue Relaxin Concentrations in the Pregnant Hamster and Fetus Using a Homologous Radioimmunoassay

Abstract A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 ± 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 ± 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 ± 0.001 μg/g tissue), increased to Day 15 (6.96 ± 0.86 μg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.

Endocrine Parameters Associated with Disruption of Parturition after Bilateral Pelvic Neurectomy

Abstract Bilateral lesions of the pelvic nerve (BLPN) result in dystocia, but the processes which control this effect are not fully understood. Plasma progesterone, relaxin, and luteinizing hormone (LH) concentrations were measured in blood samples taken in the morning (AM) and evening (PM) of Days 20–23 of gestation from rats with BLPN or sham neurectomy. Ten of 11 sham-operated control animals delivered their entire litters by Day 23 of gestation, but animals with BLPN did not complete parturition by Day 23 when they were sacrificed. Progesterone concentrations were greater in rats with BLPN than in sham-operated rats on Day 20 PM and Day 21 AM, but hormone concentrations declined to minimal values by Day 22 in both groups. Relaxin concentrations were greater in rats with BLPN than in sham-operated rats on Day 21 PM. Thereafter, relaxin concentrations decreased to reach minimum values on Day 23 in both groups. LH concentrations were low throughout the period of study in rats with BLPN; however, a postpartum LH surge was detected in all sham-operated animals. Data from this study indicate that the pelvic nerve does not control parturition by modulating serum relaxin and progesterone concentrations; however, these data suggest that impulses carried by the pelvic nerve influence ovarian secretion of these hormones. In addition, these data indicate that the pelvic nerve transmits stimuli from the cervix to the hypothalamus to facilitate the postpartum LH surge.