Relaxin increases secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 during uterine and cervical growth and remodeling in the pig.

Matrix metalloproteinases are proteolytic enzymes that degrade the extracellular matrix and are essential for tissue remodeling. Uterine and cervical growth require remodeling of structural barriers to cell invasion and matrix metalloproteinase-2 and -9 degrade type IV collagen, the major component of basement membranes. Relaxin stimulates uterine and cervical growth and remodeling, which includes remodeling of support elements such as basement membranes. The objective of this study was to determine whether relaxin alters the production and/or activity of matrix metalloproteinase-2 and -9 in the uterus or cervix of the pig. The growth-promoting effects of relaxin were elicited by administering relaxin to prepubertal gilts every 6 h for 54 h. The expression of matrix metalloproteinase-2 and matrix metalloproteinase-9 was characterized by gel zymography, and proteins were quantified by immunoblotting. Total enzyme activity was measured using matrix metalloproteinase-specific fluorescent substrate assays. In both uterine and cervical tissues, immunoreactive matrix metalloproteinase-2 and matrix metalloproteinase-9 protein expression was similar in relaxin-treated and control animals. However, tissue-associated gelatinase activity was attenuated by relaxin (P < 0.05). In contrast, relaxin significantly increased the secretion of active matrix metalloproteinase-2 and -9 protein into uterine fluid (P < 0.05). Given the importance of matrix metalloproteinases in extracellular matrix degradation, the observation that relaxin promotes uterine secretion of matrix metalloproteinase-2 and -9 supports the concept that relaxin facilitates the growth and remodeling of reproductive tissues by increasing extracellular proteolysis in the pig reproductive tract.

Expression of uterine and cervical epithelial cadherin during relaxin-induced growth in pigs.

Epithelial cadherin (E-cadherin), a member of the cadherin family of calcium-dependent adhesion molecules, is present in reproductive tissues. Relaxin, a hormone important for uterine and cervical growth in pigs, increases the expression of E-cadherin in the MCF-7 mammary epithelial cell line. The objective of this study was to characterize the expression of E-cadherin during relaxin-induced growth of the uterus and cervix in an immature pig model, independent of high circulating steroids. After administration of relaxin to prepubertal gilts, the uterus and cervix were collected. E-cadherin mRNA and protein were measured by northern and western blot analysis, respectively. A 120 kDa protein band, corresponding to E-cadherin, was detected in all tissues examined. Relaxin significantly (P < 0.05) increased the amount of E-cadherin protein in the uterus (P < 0.05), whereas no significant changes were observed in E-cadherin protein in the cervix. A 4.2 kb E-cadherin transcript was detected in all tissues and E-cadherin mRNA was significantly higher (P < 0.05) in uteri from relaxin-treated gilts compared with control gilts. E-cadherin was localized by immunocytochemistry to the epithelial cells of the uterine and cervical lumen, and the uterine glandular epithelium. Quantitative analysis revealed that administration of relaxin significantly increased (P < 0.05) the height of the uterine luminal epithelium compared with that of the controls. This is the first report of the expression of E-cadherin in the uterus and cervix of pigs. The findings from this study indicate that relaxin increases the expression of uterine E-cadherin in the reproductive tract of pigs. Administration of relaxin to prepubertal gilts in vivo increased uterine epithelial cell growth independent of circulating steroids, with a concomitant increase in E-cadherin expression.

Expression of connexin-26, -32, and -43 gap junction proteins in the porcine cervix and uterus during pregnancy and relaxin-induced growth.

Connexin (CX) proteins participate in growth, differentiation, and tissue remodeling. Relaxin-stimulated reproductive tissue growth and remodeling may be facilitated by enhanced intracellular communication. This study was an examination of the effects of relaxin in vivo on expression of CX-26, CX-32, and CX-43 in the cervix and uterus of prepubertal pigs. In addition, expression of these proteins was monitored in the sow uterus during pregnancy. Relaxin was administered to prepubertal gilts every 6 h for 54 h. CX expression was characterized by immunoblotting and localized by immunofluorescence. Significant increases in all three CXs were observed in the cervix following relaxin treatment (P < 0.05). Uterine CX proteins were also significantly higher (P < 0.05) in relaxin-treated animals compared to controls. The CX protein level in relaxin-treated animals was similar to that observed during the second half of pregnancy, but below levels found in mature, nonpregnant sows. This is the first evidence for specific CX expression in the porcine cervix, and the first study to show that relaxin increases the expression of CX proteins in the porcine uterus and cervix. The data show that CX proteins are differentially regulated in the uterus of the pig during pregnancy. These data support a role for CX-mediated communication during relaxin-induced reproductive tissue growth and remodeling.

Trophic effects of relaxin on reproductive tissue: role of the IGF system.

Although the growth promoting actions of relaxin on the reproductive tract have been well documented, the means by which relaxin stimulates reproductive tissue growth has not been identified. This report is an overview of studies from our laboratory investigating the role of the insulin-like growth factor (IGF) system in relaxin-induced growth of ovarian and uterine tissues. In the pig ovary, concentrations of relaxin that promote both theca and granulosa cell (GC) DNA synthesis in vitro also significantly (P < 0.05) increased GC IGF-I secretion. When IGF-I activity was blocked in the presence of an IGF-I antibody, the trophic effects of relaxin on GC [3H]thymidine incorporation into DNA were inhibited. However, there was no effect of relaxin on GC IGF binding proteins or IGF-I receptor. In the uterus, in vivo relaxin administration to prepubertal pigs resulted in the stimulation of growth and increases in uterine luminal IGF-I, IGF-II, and IGF binding proteins-2 and -3 secretion (P < 0.05). Thus, the trophic effects of relaxin on ovarian granulosa cells and the uterus involve tissue-specific changes in the IGF system. Additional studies are necessary to better understand the contribution of relaxin to follicular growth and uterine accommodation. These include characterization of the relaxin receptor and post-receptor binding events, as well as the potential impact of relaxin on other growth factor systems and how these systems interact to ultimately drive reproductive tissue growth.

Phosphodiesterase 4B2 is the predominant phosphodiesterase species and undergoes differential regulation of gene expression in human monocytes and neutrophils.

The type 4 phosphodiesterase (PDE4) is the predominant PDE isozyme in various leukocytes and plays a key role in the regulation of inflammatory cell activation. There are four PDE4 subtypes (A, B, C, and D), and within each subtype, there are multiple variants. Very recently, we found in monocytes that PDE4B gene expression is selectively induced by lipopolysaccharide (LPS) and that the induction is inhibited by interleukin (IL)-10 and IL-4. In this study, we show that the PDE4B gene is constitutively expressed in neutrophils and that this expression remains unaffected by LPS or IL-10. PDE4B is the predominant subtype in neutrophils and in unstimulated or LPS-stimulated monocytes, and in these cells, the PDE4B2 variant is the only detectable molecular species of PDE4B. Therefore, PDE4B2 is the predominant PDE isoform in human neutrophils and monocytes, and its expression is regulated differently by these two cell types. Furthermore, leukocytes are the most dominant source of PDE4B2, suggesting that PDE4B2 is a relatively specific target for discovering anti-inflammatory drugs.

Relaxin secretion and gene expression in porcine granulosa and theca cells are stimulated during in vitro luteinization.

During formation of the corpus luteum, the primary source of relaxin switches from theca cells (TC) to granulosa-derived, large luteal cells. What controls this shift in relaxin production is poorly understood. The objective of this study was to observe the effect of luteinization on relaxin gene expression and secretion by porcine granulosa (GC) and TC using an in vitro model. TC and GC from medium-sized porcine follicles (4-6 mm) were treated for up to 8 days with LH (250 ng/ml) and/or insulin-like growth factor-I (IGF-I; 10 ng/ml). Media were assayed for relaxin and progesterone by RIA, changes in cell morphology were recorded, and total RNA was subjected to reverse transciption-polymerase chain reaction to monitor relaxin gene expression. In vitro luteinization, induced with LH + IGF-I treatment, was confirmed in both GC and TC by a change in morphology and a sustained, significant rise in progesterone secretion. In luteinizing GC, relaxin secretion was first detected after 5 treatment days, and steadily rose until it became significantly higher (p < 0.001) by treatment Days 7-8. In contrast, relaxin release from luteinizing TC was significant after only 2 days of treatment (p < 0.05) and increased consistently over the 8-day culture period (p < 0.001). In GC, relaxin mRNA was not detected until treatment Day 4 and became significantly higher (p < 0.001) by Day 8, the final treatment day. Relaxin transcript in luteinizing TC was low on treatment Days 2-4 and significantly higher (p < 0.01) by treatment Days 6 and 8. In summary, the present study demonstrates that hormones important in the control of luteinization are essential for regulating relaxin gene expression and secretion by GC and TC in the porcine follicle.

Relaxin protein and gene expression in ovarian follicles of immature pigs.

Relaxin production by the ovarian follicle of gonadotropin-primed, prepubertal gilts is well documented. As far as we are aware, a source of relaxin in pig follicles, independent of gonadotropins, has not yet been reported. Therefore, the objective of this study was to determine whether relaxin is produced in porcine follicles in the absence of exogenous or cyclic gonadotropins. In immature pigs, immunoreactive relaxin was detected in fluids from small (1-3 mm), medium (4-5 mm) and large (>6 mm) follicles and localized to the theca interna of large follicles. Relaxin levels in follicular fluid significantly increased with follicle size (P<0.05). Relaxin mRNA was detected in whole small- and medium-sized follicles. In large follicles, the relaxin gene was expressed in thecal layers, but not granulosa cells. The abundance of relaxin transcript did not change with follicle size. In summary, relaxin protein and mRNA were detected in porcine follicles from immature animals, indicating that relaxin is produced in the porcine follicle in the absence of exogenous or cyclic gonadotropins. Relaxin's in vitro growth effects on porcine granulosa and theca cells support this follicular relaxin as a growth modulator during porcine follicular development.

Regulation of urokinase- and tissue-type plasminogen activator by relaxin in the uterus and cervix of the prepubertal gilt.

Changes in plasminogen activator are associated with the reproductive tissue remodelling that occurs during growth. Given the trophic effects of relaxin on the pig uterus and cervix, the present study was designed to examine the impact of relaxin on urokinase and tissue-type plasminogen activator (uPA and tPA) protein and activity in the uterus and cervix of prepubertal pigs. After relaxin administration in vivo to induce growth of the immature uterus and cervix, plasminogen activator activity was measured in uterine flushes and uterine and cervical tissue using a chromogenic substrate assay. Immunoreactive uPA and tPA protein in uterine flushes and uterine and cervical tissue was detected by western blotting. Urokinase plasminogen activator activity was significantly higher (P < 0.05) in uterine flushes from relaxin-treated animals than in controls. However, there was no change in uterine flush tPA activity or protein in response to in vivo relaxin treatment. There was no evidence for acid-labile inhibitors of plasminogen activator in uterine flushes of any of the animals. Cell-associated uterine tissue uPA and tPA activity, as well as protein, were similar in relaxin-treated and control prepubertal pigs. In the cervix, cell-associated tPA activity decreased significantly (P < 0.05) in relaxin-treated animals, while cervical uPA activity was unchanged. These results support the view that at least one means by which relaxin promotes pig uterine growth is by increasing uterine secretion of uPA. In addition, these studies suggest that relaxin administration in vivo to prepubertal gilts has tissue-specific effects with respect to plasminogen activator.

Relaxin increases insulin-like growth factors (IGFs) and IGF-binding proteins of the pig uterus in vivo.

Relaxin promotes growth of reproductive tissues, including the uterus. Although we have evidence of a role for insulin-like growth factor I (IGF-I) in mediating relaxin-induced growth of porcine granulosa cells in vitro, the mechanism of action by which relaxin enhances uterine growth has not been identified. To investigate a role for the uterine insulin-like growth factor (IGF) system in relaxin-induced uterine growth, we monitored the effects of relaxin on porcine IGFs and IGF-binding proteins (IGFBPs) in vivo. The trophic effects of relaxin on the uterus were elicited by administering relaxin or saline to prepubertal gilts every 6 h for 54 h. Three hours after the last injection, uterine flushes, uteri, follicular fluid, and ovaries were collected. Estradiol was measured in plasma and follicular fluid to confirm the prepubertal status of each animal. Significantly higher concentrations of uterine lumen IGF-I (P < 0.05) and IGF-II (P < 0.01) were observed in animals treated with relaxin. However, relaxin administration did not affect uterine IGF-I and -II gene expression, as determined by a ribonuclease protection assay and Northern analysis, respectively. In uterine flushes, relaxin treatment increased an IGFBP doublet (33 and 34.5 kDa) and IGFBP-3. The uterine IGFBP doublet was identified as IGFBP-2 by immunoprecipitation. Plasma or follicular fluid IGFs and IGFBPs were unaffected by relaxin administration. In addition, relaxin did not influence IGF-I binding to its uterine receptor. This is the first study to demonstrate regulation of the pig uterine IGF system by relaxin. In conclusion, the data point to IGF-I, IGF-II, IGFBP-2, and IGFBP-3 as putative mediators of relaxin-induced uterine growth in the pig.

Relaxin-induced deoxyribonucleic acid synthesis in porcine granulosa cells is mediated by insulin-like growth factor-I.

Relaxin stimulates in vitro DNA synthesis and cell proliferation of porcine granulosa cells (GC) and theca cells. The objective of the study reported here was to determine whether components of the ovarian insulin-like growth factor (IGF) system mediate relaxin's growth-promoting effects on porcine GC in vitro. In small follicle GC, relaxin (1-100 ng/ml) significantly (p < 0.05) increased IGF-I secretion to 25-34% above control. Hormonal responsiveness of GC was shown by incubation with FSH (200 ng/ml), which resulted in 125% stimulation of IGF-I secretion relative to that in cells incubated alone. When IGF-I activity in the GC cultures was neutralized with a specific IGF-I antibody, relaxin (10 and 100 ng/ml)-induced [3H]thymidine incorporation was inhibited (p < 0.05). Coincubation with IGF-I antibody also suppressed basal and IGF-I (10 ng/ml)-induced [3H]thymidine incorporation into GC DNA, but had no effect on insulin (1 microgram/ml)-induced DNA synthesis, demonstrating the specificity and lack of toxicity of the IGF-I antibody. Ligand blot analysis showed no change in secretion of GC IGF binding protein (IGFBP) in response to relaxin (1, 10, and 100 ng/ml). In contrast, IGF-I (10 ng/ml) increased secretion of IGFBP-3 and -5, whereas FSH (200 ng/ml) decreased IGFBP-3 secretion and increased IGFBP-4 secretion (p < 0.05). In IGF-I receptor competition studies, IGF-I, but not relaxin, displaced [125I]IGF-I from the GC IGF-I receptor. These studies provide direct evidence for an interaction of relaxin and the ovarian IGF system. They are the first to show 1) a stimulatory effect of relaxin on IGF-I secretion; 2) the necessity of IGF-I activity for relaxin-induced GC DNA synthesis; and 3) the absence of an effect of relaxin on GC IGFBPs or IGF-I receptor. These findings support a paracrine role for relaxin in the porcine follicle and show that relaxin acts indirectly to promote follicle growth by stimulating GC IGF-I secretion.