Ginkgolic acid improves bleomycin-induced pulmonary fibrosis by inhibiting SMAD4 SUMOylation.

Idiopathic pulmonary fibrosis (IPF) is a refractory chronic respiratory disease with progressively exacerbating symptoms and a high mortality rate. There are currently only two effective drugs for IPF; thus, there is an urgent need to develop new therapeutics. Previous experiments have shown that ginkgolic acid (GA), as a SUMO-1 inhibitor, exerted an inhibitory effect on cardiac fibrosis induced by myocardial infarction. Regarding the pathogenesis of PF, previous studies have concluded that small ubiquitin-like modifier (SUMO) polypeptides bind multiple target proteins and participate in fibrosis of multiple organs, including PF. In this study, we found altered expression of SUMO family members in lung tissues from IPF patients. GA mediated the reduced expression of SUMO1/2/3 and the overexpression of SENP1 in a PF mouse model, which improved PF phenotypes. At the same time, the protective effect of GA on PF was also confirmed in the SENP1-KO transgenic mice model. Subsequent experiments showed that SUMOylation of SMAD4 was involved in PF. It was inhibited by TGF-ß1, but GA could reverse the effects of TGF-ß1. SENP1 also inhibited the SUMOylation of SMAD4 and then participated in epithelial-mesenchymal transition (EMT) downstream of TGF-ß1. We also found that SENP1 regulation of SMAD4 SUMOylation affected reactive oxygen species (ROS) production during TGF-ß1-induced EMT and that GA prevented this oxidative stress through SENP1. Therefore, GA may inhibit the SUMOylation of SMAD4 through SENP1 and participate in TGF-ß1-mediated pulmonary EMT, all of which reduce the degree of PF. This study provided potential novel targets and a new alternative for the future clinical testing in PF.

SMAD4 transcriptionally activates GCN5 to inhibit apoptosis and promote osteogenic differentiation in dexamethasone-induced human bone marrow mesenchymal stem cells.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a serious complication caused by long-term or excessive use of glucocorticoids (GCs). General control non-derepressible 5 (GCN5) has been reported to be lowly expressed in bone tissue. Therefore, this paper attempts to investigate the role of GCN5 in SONFH and identify the potential regulatory mechanism. EXPERIMENTAL DESIGN: Following human bone mesenchymal stem cells (hBMSCs) being stimulated with dexamethasone (Dex), GCN5 expression was detected using RT-qPCR and western blotting. Then, GCN5 was overexpressed and cell viability was assessed by cell counting kit and lactate dehydrogenase kit. Cell apoptosis was determined with terminal deoxynucleotidyl transferase dUTPnickendlabeling (TUNEL) and the expression of apoptosis-related proteins was evaluated using western blotting. Alkaline phosphatase (ALP) staining and alizarin red staining were adopted for the analysis of osteogenic differentiation. Additionally, the relationship between small mothers against decapentaplegic protein 4 (SMAD4) and GCN5 was predicted by hTFtarget website and verified by luciferase reporter- and chromatin immunoprecipitation (ChIP) assays. Subsequently, SMAD4 was silenced to determine cell viability, apoptosis and osteogenic differentiation in Dex-induced hBMSCs with GCN5 upregulation. RESULTS: GCN5 expressed lower in hBMSCs exposed to Dex. GCN5 overexpression elevated cell viability, attenuated apoptosis and promoted osteogenic differentiation of hBMSCs. Additionally, SMAD4 transcriptionally activated GCN5 and upregulated GCN5 expression. While SMAD4 knockdown reversed the protective effects of GCN5 overexpression on Dex-induced cell viability loss, apoptosis increase and osteogenic differentiation inhibition in hBMSCs. CONCLUSIONS: SMAD4 transcriptionally activated GCN5 to inhibit apoptosis and promote osteogenic differentiation in Dex-induced hBMSCs.

Myostatin increases human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling†.

Placental insufficiency disorders are major obstetric complications that share a common phenomenon of poor placental trophoblast cell invasion and remodeling of uterine tissues. Myostatin is a transforming growth factor (TGF)-ß superfamily member well known for its important role in muscle growth control. Myostatin is also produced in the placenta and has been shown to regulate some trophoblast functions. However, its roles in placental development are still poorly understood. In this study, we tested the hypothesis that myostatin increases trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling. Primary and immortalized (HTR8/SVneo) trophoblast cells were used as study models. Matrigel-coated transwell invasion assays were used to study the effects of recombinant human myostatin on trophoblast cell invasion. Reverse transcription quantitative real-time polymerase chain reaction and Western blot were used to measure myostatin effects on N-cadherin mRNA and protein levels, respectively. Small inhibitor molecules as well as siRNA-mediated knockdown were used to block myostatin receptor and downstream signaling, respectively. Data were analyzed either by unpaired Student T test or one-way analysis of variance followed by Newman Keuls test for multiple group comparisons. Myostatin significantly increased primary and HTR8/SVneo trophoblast cell invasion. Moreover, myostatin upregulated N-cadherin mRNA and protein levels in a time-dependent manner in both study models. These effects were blocked by inhibition of TGF-ß type I receptors as well as siRNA-mediated knockdown of SMAD2/3 combined or common SMAD4. Importantly, myostatin-induced trophoblast cell invasion was abolished by knockdown of N-cadherin, SMAD2/3, or SMAD4. Myostatin may increase human trophoblast cell invasion by upregulating N-cadherin via SMAD2/3-SMAD4 signaling.

Kartogenin Promotes the BMSCs Chondrogenic Differentiation in Osteoarthritis by Down-Regulation of miR-145-5p Targeting Smad4 Pathway.

BACKGROUND: Transplantation of mesenchymal stem cells (MSCs) is a potential therapeutic strategy for cartilage degeneration of osteoarthritis (OA). But controlling chondrogenic differentiation of the implanted MSCs in the joints remains a challenge. The role of kartogenin (KGN) for chondrogenesis of MSCs has been widely reported, however, the mechanism of chondrogenesis has not been elucidated in OA. METHODS: In this study, we investigated the miR-145-5p, TGF-ß, Samd4, and p-stat3/stat3 expression in cartilage of OA patients and bone marrow mesenchymal stem cells (BMSCs) treated with KGN or miR-145-5p inhibitor. In addition, the cell proliferation and chondrogenic differentiation in vitro and in vivo of BMSCs treated with KGN was also detected. RESULTS: In OA patients, the expression of miR-145-5p was up-regulated, and the expression of TGF-ß, Samd4, and p-stat3/stat3 was inhibited. When the BMSCs treated with miR-145-5p inhibitor, the expression of TGF-ß, Samd4, and p-stat3/stat3 was also significantly up-regulated. KGN-treated BMSCs had better proliferation and chondrogenic differentiation by up-regulating the expression of Sox 9, Col-2a1, aggrecan in vitro and in OA by down-regulation of miR-145-5p targeting Smad4 pathway. Moreover, intra-articular injection of KGN-treated BMSCs had a better pain relief effect in OA. CONCLUSION: The double effect on cartilage protection and pain relief indicates a great potential of intra-articular injection of KGN-treated BMSCs for the treatment of OA.

Effects of Geniposide from Gardenia Fruit Pomace on Skeletal-Muscle Fibrosis.

Geniposide is the main bioactive constituent of gardenia fruit. Skeletal-muscle fibrosis is a common and irreversibly damaging process. Numerous studies have shown that geniposide could improve many chronic diseases, including metabolic syndrome and tumors. However, the effects of geniposide on skeletal-muscle fibrosis are still poorly understood. Here, we found that crude extracts of gardenia fruit pomace could significantly decrease the expression of profibrotic genes in vitro. Moreover, geniposide could also reverse profibrotic-gene expression induced by TGF-ß and Smad4, a regulator of skeletal-muscle fibrosis. In addition, geniposide treatment could significantly downregulate profibrotic-gene expression and improve skeletal-muscle injuries in a mouse model of contusion. These results together suggest that geniposide has an antifibrotic effect on skeletal muscle through the suppression of the TGF-ß-Smad4 signaling pathway.

Smad2 and Smad3 have opposing roles in breast cancer bone metastasis by differentially affecting tumor angiogenesis.

Transforming growth factor (TGF)-beta can suppress and promote breast cancer progression. How TGF-beta elicits these dichotomous functions and which roles the principle intracellular effector proteins Smad2 and Smad3 have therein, is unclear. Here, we investigated the specific functions of Smad2 and Smad3 in TGF-beta-induced responses in breast cancer cells in vitro and in a mouse model for breast cancer metastasis. We stably knocked down Smad2 or Smad3 expression in MDA-MB-231 breast cancer cells. The TGF-beta-induced Smad3-mediated transcriptional response was mitigated and enhanced by Smad3 and Smad2 knockdown, respectively. This response was also seen for TGF-beta-induced vascular endothelial growth factor (VEGF) expression. TGF-beta induction of key target genes involved in bone metastasis, were found to be dependent on Smad3 but not Smad2. Strikingly, whereas knockdown of Smad3 in MDA-MB-231 resulted in prolonged latency and delayed growth of bone metastasis, Smad2 knockdown resulted in a more aggressive phenotype compared with control MDA-MB-231 cells. Consistent with differential effects of Smad knockdown on TGF-beta-induced VEGF expression, these opposing effects of Smad2 versus Smad3 could be directly correlated with divergence in the regulation of tumor angiogenesis in vivo. Thus, Smad2 and Smad3 differentially affect breast cancer bone metastasis formation in vivo.