Osthole exhibits the remedial potential for polycystic ovary syndrome mice through Nrf2-Foxo1-GSH-NF-κB pathway.

Polycystic ovary syndrome (PCOS) is the primary cause of female infertility with a lack of universal therapeutic regimen. Although osthole exhibits numerous pharmacological activities in treating various diseases, its therapeutic effect on PCOS is undiscovered. The present study found that application of osthole improved the symptoms of PCOS mice through preventing ovarian granulosa cells (GCs) production of more estrogen and alleviating the liberation of pro-inflammatory cytokine interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha. Meanwhile, osthole enhanced ovarian antioxidant capacity and alleviated intracellular reactive oxygen species (ROS) accumulation with a concurrent attenuation for oxidative stress, while intervention of antioxidant enzymic activity and glutathione (GSH) synthesis neutralized the salvation of osthole on GCs secretory disorder and chronic inflammation. Further analysis revealed that osthole restored the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and forkhead box O 1 (Foxo1) whose repression antagonized the amelioration of osthole on the insufficiency of antioxidant capacity and accumulation of ROS. Moreover, Nrf2 served as an intermedium to mediate the regulation of osthole on Foxo1. Additionally, osthole restricted the phosphorylation of IκBα and nuclear factor kappa B (NF-κB) subunit p65 by DHEA and weakened the transcriptional activity of NF-κB, but this effectiveness was abrogated by the obstruction of Nrf2 and Foxo1, whereas adjunction of GSH renewed the redemptive effect of osthole on NF-κB whose activation caused an invalidation of osthole in rescuing the aberration of GCs secretory function and inflammation response. Collectively, osthole might relieve the symptoms of PCOS mice via Nrf2-Foxo1-GSH-NF-κB pathway.

Melatonin modulates endometrial decidualization via NOTCH1-NRF2-FOXO1-GSH pathway†.

Melatonin is important for oocyte maturation, fertilization, early embryonic development, and embryo implantation, but less knowledge is available regarding its role in decidualization. The present study found that melatonin did not alter the proliferation of human endometrial stromal cells (ESCs), as well as cell cycle progress, but suppressed stromal differentiation after binding to the melatonin receptor 1B (MTNR1B), which was visualized in decidualizing ESCs. Further analysis evidenced that application of melatonin resulted in the diminishment for NOTCH1 and RBPJ expression. Supplementation of recombinant NOTCH1 protein (rNOTCH1) counteracted the impairment of stromal differentiation conferred by melatonin, while the addition of the NOTCH signaling pathway inhibitor DAPT aggravated the differentiation progress. Meanwhile, melatonin might restrain the expression and transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2), whose blockage accelerated the fault of stromal differentiation under the context of melatonin, but this restraint was subsequently ameliorated by rNOTCH1. Forkhead box O 1 (FOXO1) was identified as a downstream target of melatonin in decidualization. Repression of NRF2 antagonized the retrieval of rNOTCH1 due to aberrant FOXO1 expression elicited by melatonin. Moreover, melatonin brought about the occurrence of oxidative stress accompanied by an obvious accumulation of intracellular reactive oxygen species and a significant reduction in glutathione (GSH) content, as well as enzymatic activities of glutathione peroxidase and glutathione reductase, whereas supplementation of rNOTCH1 improved the above-mentioned effects. Nevertheless, this improvement was disrupted by the blockage of NRF2 and FOXO1. Furthermore, addition of GSH rescued the defect of stromal differentiation by melatonin. Collectively, melatonin might impair endometrial decidualization by restraining the differentiation of ESCs dependent on NOTCH1-NRF2-FOXO1-GSH pathway after binding to the MTNR1B receptor.

HB-EGF induces mitochondrial dysfunction via estrogen hypersecretion in granulosa cells dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway.

Polycystic ovarian syndrome (PCOS) is one of the most prevalent endocrinopathies and the leading cause of anovulatory infertility, but its pathogenesis remains elusive. Although HB-EGF is involved in ovarian cancer progression, there is still no clarity about its relevance with PCOS. The present study exhibited that abundant HB-EGF was noted in follicular fluid from PCOS women, where it might induce the granulosa cells (GCs) production of more estrogen via the elevation of CYP19A1 expression after binding to EGFR. Furthermore, HB-EGF transduced intracellular downstream cAMP-PKA signaling to promote the phosphorylation of JNK and ERK whose blockage impeded the induction of HB-EGF on estrogen secretion. Meanwhile, HB-EGF enhanced the accumulation of intracellular Ca2+ whose chelation by BAPTA-AM abrogated the stimulation of HB-EGF on FOXO1 along with an obvious diminishment for estrogen production. cAMP-PKA-JNK/ERK-Ca2+ pathway played an important role in the crosstalk between HB-EGF and FOXO1. Treatment of GCs with HB-EGF resulted in mitochondrial dysfunction as evinced by the reduction of ATP content, mtDNA copy number and mitochondrial membrane potential. Additionally, HB-EGF facilitated the opening of mitochondrial permeability transition pore via targeting BAX and raised the release of cytochrome C from mitochondria into the cytosol to trigger the apoptosis of GCs, but this effectiveness was counteracted by estrogen receptor antagonist. Collectively, HB-EGF might induce mitochondrial dysfunction and GCs apoptosis through advancing estrogen hypersecretion dependent on cAMP-PKA-JNK/ERK-Ca2+-FOXO1 pathway and act as a promising therapeutic target for PCOS.

TAZ as a novel regulator of oxidative damage in decidualization via Nrf2/ARE/Foxo1 pathway.

TAZ, as a crucial effector of Hippo pathway, is required for spermatogenesis and fertilization, but little is known regarding its physiological function in uterine decidualization. In this study, we showed that TAZ was localized in the decidua, where it promoted stromal cell proliferation followed by accelerated G1/S phase transition via Ccnd3 and Cdk4 and induced the expression or activity of stromal differentiation markers Prl8a2, Prl3c1 and ALP, indicating the importance of TAZ in decidualization. Knockdown of TAZ impeded HB-EGF induction of stromal cell proliferation and differentiation. Under oxidative stress, TAZ protected stromal differentiation against oxidative damage by reducing intracellular ROS and enhancing cellular antioxidant capacity dependent on the Nrf2/ARE/Foxo1 pathway. TAZ strengthened the transcriptional activity of Nrf2 which directly bound to the antioxidant response element (ARE) of Foxo1 promoter region. Additionally, silencing TAZ caused accumulation of intracellular ROS through heightening NOX activity whose blockade by APO reversed the disruption in stromal differentiation. Further analysis revealed that TAZ might restore mitochondrial function, as indicated by the increase in ATP level, mtDNA copy number and mitochondrial membrane potential with the reduction in mitochondrial superoxide. Additionally, TAZ modulated the activities of mitochondrial respiratory chain complexes I and III whose suppression by ROT and AA resulted in the inability of TAZ to defend against oxidative damage to stromal differentiation. Moreover, TAZ prevented stromal cell apoptosis by upregulating Bcl2 expression and inhibiting Casp3 activity and Bax expression. In summary, TAZ might mediate HB-EGF function in uterine decidualization through Ccnd3 and ameliorate oxidative damage to stromal cell differentiation via Nrf2/ARE/Foxo1 pathway.

Genistein exhibits therapeutic potential for PCOS mice via the ER-Nrf2-Foxo1-ROS pathway.

Polycystic ovarian syndrome (PCOS) is a complex endocrinopathy in women of reproductive age and the main cause of female infertility, but there is no universal drug for PCOS therapy. As a predominant dietary isoflavone present in soybeans, genistein (GEN) possesses estrogenic and antioxidative properties, but limited information is available regarding its therapeutic potential and underlying molecular mechanism in PCOS. In this study, we found that GEN might restore the estrous cycle of PCOS mice and ameliorate the elevation of circulating T, AMH and LH levels as well as LH/FSH ratios along with reduced cystic follicles, indicating the importance of GEN in PCOS therapy. Meanwhile, GEN improved the ovarian secretion function of PCOS mice and attenuated oxidative damage of the ovary through enhancing its antioxidant capability dependent on ER. Supplementation of GEN improved the defect of the ATP level and mitochondrial membrane potential, indicating the significance of GEN in preventing mitochondrial dysfunction. Further analysis demonstrated that GEN via ER heightened the expression of Nrf2 and Foxo1 whose blockage antagonized the defence of GEN on the secretory and mitochondrial functions of ovarian granulosa cells followed by the limited antioxidant capability and increased intracellular ROS level. Moreover, nuclear translocation and transcriptional activity of Nrf2 presented a notable enhancement after exposure to GEN. Addition of the Nrf2 inhibitor ML385 hampered the GEN induction of Foxo1. Nrf2 might directly bind to the antioxidant response element of the Foxo1 promoter region. Collectively, GEN might exhibit therapeutic potential for PCOS mice via the ER-Nrf2-Foxo1-ROS pathway.

Bmp2 regulates Serpinb6b expression via cAMP/PKA/Wnt4 pathway during uterine decidualization.

Serpinb6b is a novel member of Serpinb family and found in germ and somatic cells of mouse gonads, but its physiological function in uterine decidualization remains unclear. The present study revealed that abundant Serpinb6b was noted in decidual cells, and advanced the proliferation and differentiation of stromal cells, indicating a creative role of Serpinb6b in uterine decidualization. Further analysis found that Serpinb6b modulated the expression of Mmp2 and Mmp9. Meanwhile, Serpinb6b was identified as a target of Bmp2 regulation in stromal differentiation. Treatment with rBmp2 resulted in an accumulation of intracellular cAMP level whose function in this differentiation program was mediated by Serpinb6b. Addition of PKA inhibitor H89 impeded the Bmp2 induction of Serpinb6b, whereas 8-Br-cAMP rescued the defect of Serpinb6b expression elicited by Bmp2 knock-down. Attenuation of Serpinb6b greatly reduced the induction of constitutive Wnt4 activation on stromal cell differentiation. By contrast, overexpression of Serpinb6b prevented this inhibition of differentiation process by Wnt4 siRNA. Moreover, blockage of Wnt4 abrogated the up-regulation of cAMP on Serpinb6b. Collectively, Serpinb6b mediates uterine decidualization via Mmp2/9 in response to Bmp2/cAMP/PKA/Wnt4 pathway.

Malic enzyme 1 is important for uterine decidualization in response to progesterone/cAMP/PKA/HB-EGF pathway.

Malic enzyme 1 (Me1), a member of the malic enzymes involving in glycolytic pathway and citric acid cycle, is essential for the energy metabolism and maintenance of intracellular redox balance state, but its physiological role and regulatory mechanism in the uterine decidualization are still unknown. Current study showed that Me1 was strongly expressed in decidual cells, and could promote the proliferation and differentiation of stromal cells followed by an accelerated cell cycle transition, indicating an importance of Me1 in the uterine decidualization. Silencing of Me1 attenuated NADPH generation and reduced GR activity, while addition of NADPH improved the defect of GR activity elicited by Me1 depletion. Further analysis found that Me1 modulated intracellular GSH content via GR. Meanwhile, Me1 played a role in maintaining mitochondrial function as indicated by these observations that blockadge of Me1 led to the accumulation of mitochondrial O2- level and decreased ATP production and mtDNA copy numbers accompanied with defective mitochondrial membrane potential. In uterine stromal cells, progesterone induced Me1 expression through PR-cAMP-PKA pathway. Knockdown of HB-EGF might impede the regulation of progesterone and cAMP on Me1. Collectively, Me1 is essential for uterine decidualization in response to progesterone/cAMP/PKA/HB-EGF pathway and plays an important role in preventing mitochondrial dysfunction.

Novel insights into Dhh signaling in antler chondrocyte proliferation and differentiation: Involvement of Foxa.

The desert hedgehog (Dhh) is crucial for spermatogenesis and Leydig cell differentiation, but little is known regarding its physiological function in cartilage. In this study, Dhh mRNA was abundant in antler chondrocytes, where it advanced cell proliferation concomitant with accelerated transition from the G1 to the S phase and induced elevation of the hypertrophic chondrocyte markers, Col X and Runx2. Silencing of Ptch1 resulted in appreciable Smo accumulation and enhanced rDhh stimulation of Smo, whose impediment by cyclopamine obscured the proliferative function of Dhh and alleviated its guidance of chondrocyte differentiation. Further analysis evidenced the noteworthy positive action of Smo in the bridging between Dhh and Gli transcription factors. Obstruction of Gli1 by GANT58 caused the failed stimulation of Col X and Runx2 by rDhh. Analogously, siRNA against Gli1-3 hindered chondrocyte differentiation in the context of rDhh. Simultaneously, Gli transcription factors mediated the regulation of Dhh on Foxa1, Foxa2, and Foxa3, whose knockdown impaired chondrocyte differentiation. Attenuation of Foxa antagonized the augmentation of Col X and Runx2 generated by rDhh. Collectively, Dhh signaling through its target Foxa appears to induce antler chondrocyte proliferation and differentiation.

Genistein protects ovarian granulosa cells from oxidative stress via cAMP-PKA signaling.

Genistein is an isoflavone that has estrogen (E2 )-like activity and is beneficial for follicular development, but little is known regarding its function in oxidative stress (OS)-mediated granulosa cell (GC) injury. Here, we found that after exposure to H2 O2 , Genistein weakened the elevated levels of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA), which were regarded as the biomarkers for OS, and rescued glutathione (GSH) content and GSH/GSSG ratio accompanying with a simultaneous increase in cyclic adenosine monophosphate (cAMP) level, whereas addition of protein kinase A (PKA) inhibitor H89 impeded the effects of Genistein on the levels of ROS and MDA. Further analysis evidenced that Genistein enhanced the activities of antioxidant enzymes superoxide dismutase (SOD), GSH-peroxidase (GSH-Px), and catalase (CAT) in H2 O2 -treated GCs, but this enhancement was attenuated by H89. Under OS, Genistein improved cell viability and lessened the apoptotic rate of GCs along with a reduction in the activity of Casp3 and levels of Bax and Bad messenger RNA (mRNA), while H89 reversed the above effects. Moreover, Genistein treatment caused an obvious elevation in mitochondrial membrane potential (MMP) followed by a decline in the levels of intracellular mitochondrial superoxide, but H89 inhibited the regulation of Genistein on MMP and mitochondrial superoxide. Supplementation of Genistein promoted the secretion of E2 and increased the expression of Star and Cyp19a1 mRNA, whereas suppressed the level of progesterone (P4 ) accompanied with a decline in the level of Hsd3b1 mRNA expression. H89 blocked the regulation of Genistein on the secretion of E2 and P4 , and alleviated the ascending of Star and Cyp19a1 elicited by Genistein. Collectively, Genistein protects GCs from OS via cAMP-PKA signaling.

Hydroxysafflor yellow A exerts beneficial effects by restoring hormone secretion and alleviating oxidative stress in polycystic ovary syndrome mice.

NEW FINDINGS: What is the central question of this study? What are the potential therapeutic roles of ginsenoside Rb1 and hydroxysafflor yellow A (HSYA) in polycystic ovary syndrome (PCOS). What is the main finding and its importance? HSYA restored the oestrous cycles of PCOS mice, reduced follicular cysts in ovaries and rescued abnormal hormone secretion; ginsenoside Rb1 did not ameliorate the main symptoms of PCOS mice. HSYA alleviated oxidative stress along with an enhancement of antioxidant enzyme activity. This highlights a potential role of HSYA in PCOS therapy. ABSTRACT: Polycystic ovary syndrome (PCOS) is the most common endocrine disease resulting in female infertility. Hydroxysafflor yellow A (HSYA) and ginsenoside Rb1 have been shown to have antioxidant properties, but little is known about their impact in PCOS. Here dehydroepiandrosterone was used to induce PCOS in a mouse model that was characterized by an irregular oestrous cycle, cystic follicles and an elevated serum testosterone level. Supplementation of HSYA restored the oestrous cycle of PCOS mice, reduced follicular cysts in PCOS mouse ovaries and brought about a decline in serum testosterone level, while ginsenoside Rb1 did not ameliorate the above symptoms of PCOS mice. After HSYA treatment, there was elevation of serum oestradiol, progesterone, luteinizing hormone and anti-Müllerian hormone levels and a reduction of follicle-stimulating hormone level, but ginsenoside Rb1 only rescued the levels of follicle-stimulating hormone and anti-Müllerian hormone. Further analysis evidenced that HSYA reversed the expression of steroid hormone secretion-related genes Star, Hsd3b1, Cyp11a1 and Cyp19a1. In PCOS mice HSYA weakened the elevation of ovarian malondialdehyde, which is regarded as a biomarker for oxidative stress. Moreover, HSYA improved reduced glutathione content accompanied by a simultaneous increase in reduced to oxidized glutathione ratio, and enhanced the activities of the antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase. Collectively, HSYA exerted beneficial effects on PCOS mice by restoring hormone secretion and alleviating oxidative stress.

Hmgb3 Induces the Differentiation of Uterine Stromal Cells Through Targeting Ptn.

Uterine decidualization is crucial for placenta formation and pregnancy maintenance. Although previous studies have reported that high mobility group box 3 (Hmgb3) is involved in the regulation of cellular proliferation and differentiation, little is known regarding its physiological role in uterine decidualization. Here, in situ hybridization result exhibited a dynamic expression pattern of Hmgb3 messenger RNA (mRNA) during early gestation, and it was mainly localized to the decidua on days 6 to 8 of gestation. Consistently, elevated Hmgb3 expression was noted in the decidualizing stromal cells after intraluminal oil infusion. In uterine luminal epithelium of ovariectomized mice, estrogen induced the accumulation of Hmgb3 mRNA, which was dependent on the existence of implanting blastocyst. Simultaneously, Hmgb3 could stimulate the proliferation of uterine stromal cells and promote the expression of Prl8a2, a reliable marker for stromal cell differentiation. Further analysis evidenced that Hmgb3 might modulate the expression of pleiotropin (Ptn) in uterine stromal cells. Moreover, silencing of Ptn could impede the upregulation of Prl8a2 elicited by Hmgb3 overexpression, while overexpression of Ptn reversed the repressive effects of Hmgb3 siRNA on Prl8a2 expression. Collectively, Hmgb3 may direct uterine decidualization through targeting Ptn.

High Mobility Group Box 1 Regulates Uterine Decidualization through Bone Morphogenetic Protein 2 and Plays a Role in Kruppel-Like Factor 5-Induced Stromal Differentiation.

BACKGROUND/AIMS: High mobility group box 1 (Hmgb1) is associated with a variety of physiological processes including embryonic development, cell proliferation and differentiation, but little information is available regarding its biological role in decidualization. METHODS: In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to analyze the spatiotemporal expression of Hmgb1 in mouse uterus during the pre-implantation period, and explore its function and regulatory mechanisms during uterine decidualization. RESULTS: Hmgb1 mRNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy, but its expression was scarcely detected on day 4 of pregnancy. With the onset of embryo implantation, abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites. Meanwhile, the accumulation of Hmgb1 mRNA was visualized in the decidual cells. Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8, subfamily a, member 2 (Prl8a2), a reliable differentiation marker for decidualization. In uterine stromal cells, cAMP analogue 8-Br-cAMP up-regulated the expression of Hmgb1, but the up-regulation was abrogated by protein kinase A (PKA) inhibitor H89. Silencing of Hmgb1 by specific siRNA impeded the induction of 8-Br-cAMP on Prl8a2. Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5 (Klf5) function in stromal differentiation. Knockdown of bone morphogenetic protein 2 (Bmp2) prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression, whereas addition of exogenous recombinant Bmp2 protein (rBmp2) reversed the repression of Hmgb1 siRNA on Prl8a2 expression. CONCLUSION: Hmgb1 may play an important role during mouse uterine decidualization.

WNT4 acts downstream of BMP2 to mediate the regulation of ATRA signaling on RUNX1 expression: Implications for terminal differentiation of antler chondrocytes.

Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well-known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4, and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1.

Ptn functions downstream of C/EBPß to mediate the effects of cAMP on uterine stromal cell differentiation through targeting Hand2 in response to progesterone.

Ptn is a pleiotropic growth factor involving in the regulation of cellular proliferation and differentiation, but its biological function in uterine decidualization remains unknown. Here, we showed that Ptn was highly expressed in the decidual cells, and could induce the proliferation of uterine stromal cells and expression of Prl8a2 and Prl3c1 which were two well-established differentiation markers for decidualization, suggesting an important role of Ptn in decidualization. In the uterine stromal cells, progesterone stimulated the expression of Ptn accompanied with an accumulation of intracellular cAMP level. Silencing of Ptn impeded the induction of progesterone and cAMP on the differentiation of uterine stromal cells. Administration of PKA inhibitor H89 resulted in a blockage of progesterone on Ptn expression. Further analysis evidenced that regulation of progesterone and cAMP on Ptn was mediated by C/EBPß. During in vitro decidualization, knockdown of Ptn could weaken the up-regulation of Prl8a2 and Prl3c1 elicited by C/EBPß overexpression, while constitutive activation of Ptn reversed the repressive effects of C/EBPß siRNA on the expression of Prl8a2 and Prl3c1. Meanwhile, Ptn might mediate the regulation of C/EBPß on Hand2 which was a downstream target of Ptn in the differentiation of uterine stromal cells. Attenuation of Ptn or C/EBPß by specific siRNA blocked the stimulation of Hand2 by progesterone and cAMP. Collectively, Ptn may play a vital role in the progesterone-induced decidualization pathway.

ATRA Signaling Regulates the Expression of COL9A1 through BMP2-WNT4-RUNX1 Pathway in Antler Chondrocytes.

Although all-trans retinoic acid (ATRA) is involved in the regulation of cartilage growth and development, its regulatory mechanisms remain unknown. Here, we showed that ATRA could induce the expression of COL9A1 in antler chondrocytes. Silencing of cellular retinoic acid binding protein 2 (CRABP2) could impede the ATRA-induced upregulation of COL9A1, whereas overexpression of CRABP2 presented the opposite effect. RARα agonist Am80 induced the expression of COL9A1, whereas treatment with RARα antagonist Ro 41-5253 or RXRα small-interfering RNA (siRNA) caused an obvious blockage of ATRA on COL9A1. In antler chondrocytes, CYP26A1 and CYP26B1 weakened the sensitivity of ATRA to COL9A1. Simultaneously, Bone morphogenetic protein 2 (BMP2) and WNT4 mediated the regulation of ATRA on COL9A1 expression. Knockdown of WNT4 could abrogate the inhibitory effect of BMP2 overexpression on COL9A1. Conversely, constitutive expression of WNT4 reversed the upregulation of COL9A1 elicited by BMP2 siRNA. Together these data indicated that WNT4 might act downstream of BMP2 to mediate the effect of ATRA on COL9A1 expression. Further analysis evidenced that attenuation of runt-related transcription factor 1 (RUNX1) could prevent the stimulation of ATRA on COL9A1 expression, while exogenous rRUNX1 further enhanced this effectiveness. Moreover, RUNX1 might serve as an intermediate to mediate the regulation of BMP2 and WNT4 on COL9A1 expression. Collectively, ATRA signaling might regulate the expression of COL9A1 through BMP2-WNT4-RUNX1 pathway.

Gja1 acts downstream of Acvr1 to regulate uterine decidualization via Hand2 in mice.

Although Gja1 has been proved to play an important role in uterine decidualization, its regulatory mechanism remains largely unknown. Here, we showed that Gja1 was highly expressed in the decidual cells and promoted the proliferation of uterine stromal cells and expression of Prl8a2 and Prl3c1, which were two well-known differentiation markers for decidualization. Further analysis revealed that Gja1 might act downstream of Acvr1 and cAMP to regulate the differentiation of uterine stromal cells. Administration of cAMP analog 8-Br-cAMP to Acvr1 siRNA-transfected stromal cells resulted in an obvious increase of Gja1 expression, whereas PKA inhibitor H89 impeded the induction of Gja1 elicited by Acvr1 overexpression, indicating that cAMP-PKA signal mediates the regulation of Acvr1 on Gja1 expression. In uterine stromal cells, knockdown of Gja1 blocked the cAMP induction of Hand2 Moreover, siRNA-mediated downregulation of Hand2 impaired the stimulatory effects of Gja1 overexpression on the expression of Prl8a2 and Prl3c1, whereas constitutive expression of Hand2 reversed the inhibitory effects of Gja1 siRNA on stromal differentiation. Meanwhile, Gja1 might play a vital role in the crosstalk between Acvr1 and Hand2 Collectively, Gja1 may act downstream of cAMP-PKA signal to mediate the effects of Acvr1 on the differentiation of uterine stromal cells through targeting Hand2.

IGF1 regulates RUNX1 expression via IRS1/2: Implications for antler chondrocyte differentiation.

Although IGF1 is important for the proliferation and differentiation of chondrocytes, its underlying molecular mechanism is still unknown. Here we addressed the physiologic function of IGF1 in antler cartilage and explored the interplay of IGF1, IRS1/2 and RUNX1 in chondrocyte differentiation. The results showed that IGF1 was highly expressed in antler chondrocytes. Exogenous rIGF1 could increase the proliferation of chondrocytes and cell proportion in the S phase, whereas IGF1R inhibitor PQ401 abrogated the induction by rIGF1. Simultaneously, IGF1 could stimulate the expression of IHH which was a well-known marker for prehypertrophic chondrocytes. Further analysis evidenced that IGF1 regulated the expression of IRS1/2 whose silencing resulted in a rise of IHH mRNA levels, but the regulation was impeded by PQ401. Knockdown of IRS1 or IRS2 with specific siRNA could greatly enhance rIGF1-induced chondrocyte differentiation and reduce the expression of RUNX1. Extraneous rRUNX1 might rescue the effects of IRS1 or IRS2 siRNA on the differentiation. In antler chondrocytes, IGF1 played a role in modulating the expression of RUNX1 through IGF1R. Moreover, attenuation of RUNX1 expression advanced the differentiation elicited by rIGF1, while administration of rRUNX1 to chondrocytes treated with IGF1 siRNA or PQ401 reduced their differentiation. Additionally, siRNA-mediated downregulation of IRS1 or IRS2 in the chondrocytes impaired the interaction between IGF1 and RUNX1. Collectively, IGF1 could promote the proliferation and differentiation of antler chondrocytes. Furthermore, IRS1/2 might act downstream of IGF1 to regulate chondrocyte differentiation through targeting RUNX1.

13cRA regulates the differentiation of antler chondrocytes through targeting Runx3.

Although 13cRA is involved in the regulation of cellular proliferation and differentiation, its physiological roles in chondrocyte proliferation and differentiation still remain unknown. Here, we showed that 13cRA could induce the proliferation of sika deer antler chondrocytes and expression of Ccnd3 and Cdk6. Administration of 13cRA to antler chondrocytes resulted in an obvious increase in the expression of chondrocyte marker Col II and hypertrophic chondrocyte marker Col X. Silencing of Crabp2 expression by specific siRNA could prevent the 13cRA-induced up-regulation of Col X, whereas overexpression of Crabp2 showed the opposite effects. Further study found that Crabp2 mediated the regulation of 13cRA on the expression of Runx3 which was highly expressed in the antler cartilage and inhibited the differentiation of antler chondrocytes. Moreover, attenuation of Runx3 expression greatly raised 13cRA-induced chondrocyte differentiation. Simultaneously, 13cRA could stimulate the expression of Cyp26a1 and Cyp26b1 in the antler chondrocytes. Inhibition of Cyp26a1 and/or Cyp26b1 reinforced the effects of 13cRA on the expression of Col X and Runx3, while overexpression of Cyp26b1 rendered the antler chondrocytes hyposensitive to 13cRA. Collectively, 13cRA may play an important role in the differentiation of antler chondrocytes through targeting Runx3. Crabp2 enhances the effects of 13cRA on chondrocyte differentiation, while Cyp26a1 and Cyp26b1 weaken the sensitivity of antler chondrocytes to 13cRA.

Expression and regulation of Angiopoietins and their receptor Tie-2 in sika deer antler.

The cartilage vascularization and chondrocyte survival are essential for endochondral ossification which occurs in the process of antler growth. Angiopoietins (Ang) is a family of major angiogenic growth factors and involved in regulating the vascularization. However, the expression and regulation of Angs in the antler are still unknown. The aim of this study is to localize the expression of Ang-1, Ang-2 and their receptor Tie-2 in sika deer antler using in situ hybridization and focused on analyzing the regulation of testosterone, estrogen, all-trans-retinoic acid (ATRA) and 9cRA on their expression in antler chondrocytes. The results showed that Ang-1, Ang-2 and Tie-2 were highly expressed in antler chondrocytes. Administration of testosterone to antler chondrocytes led to a notable increase in the expression of Ang-1 and Tie-2, and a reduction in the expression of Ang-2. The similar result was also observed after estrogen treatment. In contrast, ATRA and 9cRA could inhibit the expression of Ang-1 in antler chondrocytes and heighten the expression of Ang-2. Simultaneously, ATRA could downregulate the expression of Tie-2 in antler chondrocytes at 12 and 24 h, while 9cRA upregulate the expression of Tie-2 at 3 and 6 h. Collectively, Ang-1, Ang-2 and Tie-2 are expressed in antler chondrocytes and their expression can be affected by testosterone, estrogen, ATRA and 9cRA.

Hmgn5 functions downstream of Hoxa10 to regulate uterine decidualization in mice.

Although Hmgn5 is involved in the regulation of cellular proliferation and differentiation, its physiological function during decidualization is still unknown. Here we showed that Hmgn5 was highly expressed in the decidual cells. Silencing of Hmgn5 expression by specific siRNA reduced the proliferation of uterine stromal cells and expression of Ccnd3 and Cdk4 in the absence or presence of estrogen and progesterone, whereas overexpression of Hmgn5 exhibited the opposite effects. Simultaneously, Hmgn5 might induce the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP and progesterone could up-regulate the expression of Hmgn5, but the up-regulation was impeded by H89 and RU486, respectively. Attenuation of Hmgn5 expression could block the differentiation of uterine stromal cells in response to cAMP and progesterone. Further studies found that regulation of cAMP and progesterone on Hmgn5 expression was mediated by Hoxa10. During in vitro decidualization, knockdown of Hmgn5 could abrogate Hoxa10-induced upregulation of Prl8a2 and Prl3c1, while overexpression of Hmgn5 reversed the inhibitory effects of Hoxa10 siRNA on the expression of Prl8a2 and Prl3c1. In the stromal cells undergoing decidualization, Hmgn5 might act downstream of Hoxa10 to regulate the expression of Cox-2, Vegf and Mmp2. Collectively, Hmgn5 may play an important role during mouse decidualization.