In vivo and in vitro studies of MUC1 regulation in sheep endometrium

In this study, we investigated the expression of mucin 1 (MUC1) mRNA and protein in sheep endometrium at different time points during follicular and luteal phases of estrous cycle, and also determined the effect of steroid hormone treatments and interferon tau (IFNτ) on MUC1 mRNA expression in endometrial cell culture in vitro. In experiment one, 15 Welsh mountain ewes were synchronized to a common estrus and killed at precise stages of estrous cycle corresponding to (1) pre-LH peak, (2) LH peak, (3) post-LH peak, (4) early luteal, and (5) mid-luteal. Reproductive tracts were harvested and mRNA was extracted from the endometrial tissues. Parts of the uterine horns were fixed for immunohistochemistry. In experiment two, mixed populations of ovine endometrial cells (from slaughterhouse material collected at the postovulatory stage of the estrous cycle) were cultured to 70% confluence before treatment with (1) progesterone (P₄, 10 ng/mL, for 48 hours), (2) estradiol (E₂, 100 pg/mL, for 48 hours), or with (3) E₂ priming for 12 hours (100 pg/mL) followed by P₄ (10 ng/mL) for 36 hours. These were compared with: (4) IFNτ (10 ng/mL, for 48 hours), and (5) basic medium (Dulbecco Modified Eagle Medium /F12) as control. The results showed that MUC1 mRNA and protein expression in sheep endometrium were highest during the midluteal stage and very low during the post-LH period compared with the other stages (P < 0.05). MUC1 immunostaining in the luminal epithelium was apically restricted and was not significantly different across all stages of estrous cycle except at the post-LH peak where it was significantly low. In cell culture, MUC1 mRNA expression was significantly upregulated by both steroids either singly or in combination (P < 0.05), and downregulated in the presence of IFNτ. In conclusion, endometrial MUC1 expression is cyclically regulated by both E₂ and P₄ in vivo and in vitro, and directly downregulated by IFNτ treatment in vitro.     

MMP and TIMP temporal gene expression during osteocytogenesis

Osteocytes within bone differentiate from osteoblast precursors which reside in a mineralised extracellular matrix (ECM). Fully differentiated osteocytes are critical for bone development and function but the factors that regulate this differentiation process are unknown. The enzymes primarily responsible for ECM remodelling are matrix metalloproteinases (MMP); however, the expression and role of MMPs during osteocytogenesis is undefined. Here we used MLO-A5 cells to determine the temporal gene expressions of the MMP family and their endogenous inhibitors – tissue inhibitors of metalloproteinases (TIMPs) during osteocytogenesis. RT-qPCR revealed expression of 14 Mmps and 3 Timps in MLO-A5 cells. Mmp2, Mmp23 and Mmp28 were decreased concurrent with mineralisation onset (P < 0.05*). Mmp14 and Mmp19 mRNAs were also significantly increased at day 3 (P < 0.05*) before returning to baseline levels at day 6. Decreased expressions of Timp1, Timp2 and Timp3 mRNA were observed by day 6 compared to day 0 (P < 0.05*). To examine whether these changes are linked to osteocytogenesis, we determined Mmp/Timp mRNA expressions in mineralisation-limited conditions. RT-qPCR revealed that the previously observed decreases in Mmp2, Mmp23 and Mmp28 were not observed in these mineralisation-limited cultures, therefore closely linking these MMPs with osteocyte differentiation. Similarly, we found differential expression of Timp1, Timp2 and Timp3 mRNA in mineralisation-restricted cultures (P < 0.05*). In conclusion, we have identified several members of the MMP/TIMP families as regulators of ECM remodelling necessary for the acquisition of the osteocyte phenotype.     

Cell Surface-Bound TIMP3 Induces Apoptosis in Mesenchymal Cal78 Cells through Ligand-Independent Activation of Death Receptor Signaling and Blockade of Survival Pathways

Background

The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitor of metalloproteinases (TIMPs 1–4) are responsible for the physiological remodeling of the extracellular matrix (ECM). Among all TIMPs, TIMP3 appears to play a unique role since TIMP3 is a secreted protein and, unlike the other TIMP family members, is tightly bound to the ECM. Moreover TIMP3 has been shown to be able to induce apoptotic cell death. As little is known about the underlying mechanisms, we set out to investigate the pro-apoptotic effect of TIMP3 in human mesenchymal cells.

Methodology/Principal Findings

Lentiviral overexpression of TIMP3 in mesenchymal cells led to a strong dose-dependent induction of ligand-independent apoptosis as reflected by a five-fold increase in caspase 3 and 7 activity compared to control (pLenti6/V5-GW/lacZ) or uninfected cells, whereas exogenous TIMP3 failed to induce apoptosis. Concordantly, increased cleavage of death substrate PARP and the caspases 3 and 7 was observed in TIMP3 overexpressing cultures. Notably, activation of caspase-8 but not caspase-9 was observed in TIMP3-overexpressing cells, indicating a death receptor-dependent mechanism. Moreover, overexpression of TIMP3 led to a further induction of apoptosis after stimulation with TNF-alpha, FasL and TRAIL. Most interestingly, TIMP3-overexpression was associated with a decrease in phosphorylation of cRaf, extracellular signal-regulated protein kinase (Erk1/2), ribosomal S6 kinase (RSK1) and Akt and serum deprivation of TIMP3-overexpressing cells resulted in a distinct enhancement of apoptosis, pointing to an impaired signaling of serum-derived survival factors. Finally, heparinase treatment of heparan sulfate proteoglycans led to the release of TIMP3 from the surface of overexpressing cells and to a significant decrease in apoptosis indicating that the binding of TIMP3 is necessary for apoptosis induction.

Conclusion

The results demonstrate that exclusively cell surface-bound endogenous TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival signaling pathways.     

Regulation of the immunoexpression of aquaporin 9 by ovarian hormones in the rat oviductal epithelium

The volume of oviductal fluid fluctuates during the estrous cycle, suggesting that water availability is under hormonal control. It has been postulated that sex-steroid hormones may regulate aquaporin (AQP) channels involved in water movement across cell membranes. Using a functional assay (oocytes of Xenopus laevis), we demonstrated that the rat oviductal epithelium contains mRNAs coding for water channels, and we identified by RT-PCR the mRNAs for AQP5, -8, and -9, but not for AQP2 and -3. The immunoreactivity for AQP5, -8, and -9 was localized only in epithelial cells of the oviduct. The distribution of AQP5 and -8 was mainly cytoplasmic, whereas we confirmed, by confocal microscopy, that AQP9 localized to the apical plasma membrane. Staining of AQP5, -8, and -9 was lost after ovariectomy, and only AQP9 immunoreactivity was restored after estradiol and/or progesterone treatments. The recovery of AQP9 reactivity after ovariectomy correlated with increased mRNA and protein levels after treatment with estradiol alone or progesterone administration after estradiol priming. Interestingly, progesterone administration after progesterone priming also induced AQP9 expression but without a change in mRNA levels. Levels of AQP9 varied along the estrous cycle with their highest levels during proestrus and estrus. These results indicate that steroid hormones regulate AQP9 expression at the mRNA and protein level and that other ovarian signals are involved in the expression of AQP5 and -8. Thus hormonal regulation of the type and quantity of water channels in this epithelium might control water transport in the oviductal lumen. water channels; epithelial cells; estradiol; progesterone     

Genetic variation in TIMP1 but not MMPs predict excess FEV1 decline in two general population-based cohorts

Background

An imbalance in Matrix MetalloProteases (MMPs) and Tissue Inhibitors of MMPs (TIMPs) contributes to Chronic Obstructive Pulmonary Disease (COPD) development. Longitudinal studies investigating Single Nucleotide Polymorphisms (SNPs) in MMPs and TIMPs with respect to COPD development and lung function decline in the general population are lacking.

Methods

We genotyped SNPs in MMP1 (G-1607GG), MMP2 (-1306 C/T), MMP9 (3 tagging SNPs), MMP12 (A-82G and Asn357Ser) and TIMP1 (Phe124Phe and Ile158Ile) in 1390 Caucasians with multiple FEV₁ measurements from a prospective cohort study in the general population. FEV₁ decline was analyzed using linear mixed effect models adjusted for confounders. Analyses of the X-chromosomal TIMP1 gene were stratified according to sex. All significant associations were repeated in an independent general population cohort (n = 1152).

Results

MMP2 -1306 TT genotype carriers had excess FEV₁ decline (-4.0 ml/yr, p = 0.03) compared to wild type carriers. TIMP1 Ile158Ile predicted significant excess FEV₁ decline in both males and females. TIMP1 Phe124Phe predicted significant excess FEV₁ decline in males only, which was replicated (p = 0.10) in the second cohort. The MMP2 and TIMP1 Ile158Ile associations were not replicated. Although power was limited, we did not find associations with COPD development.

Conclusions

We for the first time show that TIMP1 Phe124Phe contributes to excess FEV₁ decline in two independent prospective cohorts, albeit not quite reaching conventional statistical significance in the replication cohort. SNPs in MMPs evidently do not contribute to FEV₁ decline in the general population.     

Ovarian matrix metalloproteinases are differentially regulated during the estrous cycle but not during short photoperiod induced regression in Siberian hamsters (<Emphasis Type="Italic">Phodopus sungorus</Emphasis>)

Background  

Matrix metalloproteinases (MMPs) are implicated as mediators for ovarian remodeling events, and are involved with ovarian recrudescence during seasonal breeding cycles in Siberian hamsters. However, involvement of these proteases as the photoinhibited ovary undergoes atrophy and regression had not been assessed. We hypothesized that 1) MMPs and their tissue inhibitors, the TIMPs would be present and differentially regulated during the normal estrous cycle in Siberian hamsters, and that 2) MMP/TIMP mRNA and protein levels would increase as inhibitory photoperiod induced ovarian degeneration.     

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.     

Effects of 17β-estradiol on turkey myogenic satellite cell proliferation,differentiation, and expression of glypican-1, MyoD and myogenin

The hypothesis of this study was that 17β-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10^? 9 M following 4 days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10^? 12 M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10^? 9 M and 10^? 5 M estradiol following 3 days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.     

MicroRNA-146b-5p promotes atrial fibrosis in atrial fibrillation by repressing TIMP4

Alteration of tissue inhibitors of matrix metalloproteinases (TIMP)/matrix metalloproteinases (MMP) associated with collagen upregulation has an important role in sustained atrial fibrillation (AF). The expression of miR-146b-5p, whose the targeted gene is TIMPs, is upregulated in atrial cardiomyocytes during AF. This study was to determine whether miR-146b-5p could regulate the gene expression of TIMP4 and the contribution of miRNA to atrial fibrosis in AF. Collagen synthesis was observed after miR-146b-5p transfection in human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs)-fibroblast co-culture cellular model in vitro. Furthermore, a myocardial infarction (MI) mouse model was used to confirm the protective effect of miR-146b-5p downregulation on atrial fibrosis. The expression level of miR-146b-5p was upregulated, while the expression level of TIMP4 was downregulated in the fibrotic atrium of canine with AF. miR-146b-5p transfection in hiPSC-aCMs-fibroblast co-culture cellular model increased collagen synthesis by regulating TIMP4/MMP9 mediated extracellular matrix proteins synthesis. The inhibition of miR-146b-5p expression reduced the phenotypes of cardiac fibrosis in the MI mouse model. Fibrotic marker MMP9, TGFB1 and COL1A1 were significantly downregulated, while TIMP4 was significantly upregulated (at both mRNA and protein levels) by miR-146b-5p inhibition in cardiomyocytes of MI heart. We concluded that collagen fibres were accumulated in extracellular space on miR-146b-5p overexpressed co-culture cellular model. Moreover, the cardiac fibrosis induced by MI was attenuated in antagomiR-146 treated mice by increasing the expression of TIMP4, which indicated that the inhibition of miR-146b-5p might become an effective therapeutic approach for preventing atrial fibrosis.     

MMP/TIMP expression profiles in distinct lung disease models: implications for possible future therapies

Background

There is currently a vast amount of evidence in the literature suggesting that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in the pathogenesis of inflammatory airways diseases, such as asthma and COPD. Despite this, the majority of reports only focus on single MMPs, often only in one model system. This study aimed to investigate the profile of an extensive range of MMP/TIMP levels in three different pre-clinical models of airways disease. These models each have a different and very distinct inflammatory profile, each exhibiting inflammatory characteristics that are similar to that observed in asthma or COPD. Since these models have their own characteristic pathophysiological phenotype, one would speculate that the MMP/TIMP expression profile would also be different.

Methods

With the use of designed and purchased MMP/TIMP assays, investigation of rat MMP-2, 3, 7-14 and TIMP-1-4 mRNA expression was undertaken by Real Time PCR. The three rodent models of airways disease investigated were the endotoxin model, elastase model, and the antigen model.

Results

Intriguingly, we demonstrated that despite the distinct inflammatory profile observed by each model, the MMP/TIMP expression profile is similar between the models, in that the same MMPs/TIMPs were observed to be generally increased or decreased in all three models. It could therefore be speculated that in a particular disease, it may be a complex network of MMPs, rather than an individual MMP, together with inflammatory cytokines and other mediators, that results in the distinct phenotype of inflammatory diseases, such as asthma and COPD.

Conclusion

We believe our data may provide key information necessary to understand the role of various MMPs/TIMPs in different inflammatory airway diseases, and aid the development of more selective therapeutics without the side effect profile of current broad-spectrum MMP inhibitors.     

The orphan nuclear receptor SF-1 is involved in the effect of PCBs,DDT, and DDE on the secretion of steroid hormones and oxytocin from bovine luteal cells during the estrous cycle in vitro

The orphan receptor steroidogenic factor-1 (SF-1) is involved in the regulation of ovarian steroidogenesis in cows. It is hypothesized that estrogen-like chlorinated compounds might affect SF-1, and thus impair the function of the ovary. Bovine luteal cells from the estrous cycle (Days: 1–5, 6–10, 11–15, and 16–19) were treated for 50 hours with DDT, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene, 3,3′4,4′-tetrachlorobiphenyl or 2′2′4,4′,5,5′-hexachlorobiphenyl (each at a dose of 10 ng/mL). Luteal cells were also treated with 4-(heptyloxy)phenol (1 × 10⁻⁷ M), an SF-1 agonist, and F0160 (1 × 10⁻⁶ M), an SF-1 blocker, jointly or separately. The secretion of progesterone and oxytocin and the expression of oxytocin precursor (NP-I/OT) messenger RNA were increased (P < 0.05) by all studied xenobiotics and 4-(heptyloxy)phenol, although they were inhibited (P < 0.05) by F0160. However, the xenobiotics did not affect (P > 0.05) SF-1 messenger RNA expression. In summary, SF-1 is involved in the adverse effect of chlorinated xenobiotics on the regulation of the bovine CL.     

Comparison of proteins synthesized by polarized caprine oviductal epithelial cells and oviductal explants in vitro

Our objectives were to compare proteins secreted by caprine oviductal explants and oviductal epithelial (OE) cells in vitro. Oviducts were collected from goats on Days 1 (n=5) and 5 (n=5) of the estrous cycle. Radiolabeled secretory proteins from tissue segments and cell cultures were visualized using SDS-PAGE and fluorography. After culture, media from ampulla oviduct segments collected on Days 1 and 5 of the estrous cycle contained an acidic 97 kDa protein, which was greatly reduced in culture medium obtained from infundibulum and isthmus oviduct segments. A complex of low molecular weight proteins (14-26 kDa) could be modulated by estradiol when OE cells were cultured on plastic. This complex was constitutively expressed when OE cells were cultured on Matrigel-coated filters. Polarized OE cells were also capable of compartment-specific secretion of [L-(35)S]-methionine-labeled proteins. A 45 kDa acidic protein was predominantly secreted into the apical compartment while a 66 kDa acidic protein was preferentially localized in the basal compartment. Proteins secreted by OE cells were similar to proteins secreted by tissue segments in vitro. Therefore, under well-defined culture conditions OE cells may be useful in enhancing in vitro fertilization or early embryonic development.     

Expression of progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine endometrium during the estrous cycle and the first trimester of pregnancy

Progesterone (P4) is involved in the regulation of essential reproductive functions affecting the target cells through both nuclear progesterone receptors (PGRs) and membrane progesterone receptors. The aim of this study was to determine the mRNA and protein expression for PGRMC1, PGRMC2, SERBP1 and PGR within the bovine endometrium during the estrous cycle and the first trimester of pregnancy. There were no changes in PGRMC1 and PGRMC2 mRNA and protein expression during the estrous cycle, however, mRNA levels of PGRMC1 and PGRMC2 were increased (P < 0.001) in pregnant animals. SERBP1 mRNA expression was increased (P < 0.05), while the level of this protein was decreased (P < 0.05) on days 11–16 of the estrous cycle. The expression of PGR mRNA was higher (P < 0.01) on days 17–20 compared to days 6–10 and 11–16 of the estrous cycle and pregnancy. PGR-A and PGR-B protein levels were elevated on days 1–5 and 17–20 of the estrous cycle as compared to other stages of the cycle and during pregnancy. In conclusion, our results indicate that P4 may influence endometrial cells through both genomic and nongenomic way. This mechanism may contribute to the regulation of the estrous cycle and provide protection during pregnancy.     

Fluctuation of Aquaporin 1, 5, and 9 Expression in the Pig Oviduct during the Estrous Cycle and Early Pregnancy

Thirteen mammalian aquaporin (AQPs) isoforms with a unique tissue-specific pattern of expression have been identified. To date, 11 isoforms of AQP have been reported to be expressed in female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig oviduct during different stages of the estrous cycle and early pregnancy. The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within oviductal blood vessels. In cyclic gilts, the expression of AQP1 protein did not change significantly between days 10–12 and 14–16 but increased on days 2–4 and 18–20. AQP5 was localized in smooth muscle cells and oviductal epithelial cells. The expression of AQP5 protein did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. The anti-AQP9 antibody labeled epithelial cells of the oviduct. The expression of AQP9 did not change significantly between days 10–12 and 14–16 of the estrous cycle but increased on days 2–4 and 18–20. In pregnant gilts, expression of AQP1, 5, and 9 did not change significantly in comparison with the estrous cycle. Therefore, a functional and distinctive collaboration seems to exist among diverse AQPs in water handling during the different oviductal phases in the estrous cycle and early pregnancy.