MicroRNA-221 promotes cell proliferation, migration, and differentiation by regulation of ZFPM2 in osteoblasts.

Bone fracture is a common medical condition, which may occur due to traumatic injury or disease-related conditions. Evidence suggests that microRNAs (miRNAs) can regulate osteoblast differentiation and function. In this study, we explored the effects and mechanism of miR-221 on the growth and migration of osteoblasts using MC3T3-E1 cells. The expression levels of miR-221 in the different groups were measured by qRT-PCR. Then, miR-221 mimic and inhibitor were transfected into MC3T3-E1 cells, and cell viability and migration were measured using the CCK-8 assay and the Transwell migration assay. Additionally, the expression levels of differentiation-related factors (Runx2 and Ocn) and ZFPM2 were measured by qRT-PCR. Western blot was used to measure the expression of cell cycle-related proteins, epithelial-mesenchymal transition (EMT)-related proteins, ZFPM2, and Wnt/Notch, and Smad signaling pathway proteins. miR-221 was significantly up-regulated in the patients with lumbar compression fracture (LCM) and trochanteric fracture (TF). miR-221 promoted ALP, Runx2, and OPN expressions in MC3T3-E1 cells. miR-221 overexpression significantly increased cell proliferation, migration, differentiation, and matrix mineralization, whereas suppression of miR-221 reversed these effects. Additionally, the results displayed that ZFPM2 was a direct target gene of miR-221, and overexpression of ZFPM2 reversed the promoting effects of miR-221 overexpression on osteoblasts. Mechanistic study revealed that overexpression of miR-221 inactivated the Wnt/Notch and Smad signaling pathways by regulating ZFPM2 expression. We drew the conclusions that miR-221 overexpression promoted osteoblast proliferation, migration, and differentiation by regulation of ZFPM2 expression and deactivating the Wnt/Notch and Smad signaling pathways.

Effect of bone marrow mesenchymal stem cells on the TGF-ß1/Smad signaling pathway of hepatic stellate.

This study investigated the effect of bone marrow mesenchymal stem cells (BMCs) on the transforming growth factor-ß1 (TGF-ß1)-induced activation of the Smad signaling pathway in rat hepatic stellate cells (HSCs). There were four experimental groups: 1) a blank control group, 2) a TGF-ß1 treatment group, 3) an MSC-combined group, and 4) an induced MSC-combined group. Isolation and culture of rat liver HSCs in vitro and the proliferation of HSCs in each group were detected by MTT method. The expression of α-SMA and the TGF receptors (TbRI and II) were determined by immunohistochemical staining of HSCs in all groups, while Smad2/3, Smad4, and Smad7 mRNA expressions were detected by RT-PCR for HSCs in each group. TGF-ß1 treatment significantly promoted the proliferation of HSCs (P < 0.01); it has different inhibition effects on the proliferation of HSCs in the MSC-combined group and in the induced MSC-combined group (P < 0.05). TGF-ß1 treatment also enhanced the expression of α-SMA as compared to the control group (P < 0.01). Alternatively, when compared with the pure TGF-ß1 group, the MSC-combined group and the induced MSC-combined group showed lower α-SMA expression (P < 0.05). Activation of HSCs induced by TGF-ß1, TßRI and TßRII fluorescence was (+ + +); the fluorescences of TßRI and TßRII in MSC-combined group and in induced MSC-combined group were (+ +) and (± ~ +), respectively. The expressions of TßRI and TßRII in activated HSCs induced by TGF-ß1 were significantly decreased in the MSC-combined group (P < 0.05) and in the induced MSC-combined group (P < 0.01). The expression of HSC Smad2/3 and Smad4 was reduced in the MSC-combined group (P < 0.05) and in the induced MSC-combined group (P < 0.01), as compared to the TGF-ß1 group. However, the expression of Smad7 in HSCs was upregulated in the MSC-combined group (P < 0.05) and in the induced MSC-combined group (P < 0.01). These results indicate that BMCs can inhibit the activation and proliferation of HSCs by downregulating the expression of TßRI and TßRII in the cell membrane of HSCs. Moreover, BMCs can upregulate the expression of Smad7 and downregulate the expression of Smad2/3 and Smad4 in the HSCs induced by TGF-ß1, which resulted in an inhibition of HSC activation.

Interferon-α2b and transforming growth factor-ß1 treatments on HCC cell lines: Are Wnt/ß-catenin pathway and Smads signaling connected in hepatocellular carcinoma?

Wnt/ß-catenin pathway is often dysregulated in hepatocellular carcinoma (HCC). Activated ß-catenin accumulates in the cytosol and nucleus and forms a nuclear complex with TCF/LEF factors like TCF4. Interferon-α (IFN-α) has recently been recognized to harbor therapeutic potential in prevention and treatment of HCC. Transforming Growth Factor-ß1 (TGF-ß1) is a mediator of apoptosis, exerting its effects via Smads proteins. One mode of interaction between Wnt/ß-catenin and TGF-ß1/Smads pathways is the association of Smads with ß-catenin/TCF4. In this study we analyzed the effects of IFN-α2b and TGF-ß1 treatments on Wnt/ß-catenin pathway, Smads proteins levels, ß-catenin/TCF4/Smads interaction and proliferation and apoptotic death in HepG2/C3A and Huh7 cell lines. IFN-α2b and TGF-ß1 attenuated Wnt/ß-catenin signal by decreasing ß-catenin and Frizzled7 receptor proteins contents and the interaction of ß-catenin with TCF4. Truncated ß-catenin form present in C3A cell line also diminished after treatments. Both cytokines declined Smads proteins and their interaction with TCF4. The overall cellular response to cytokines was the decrease in proliferation and increase in apoptotic death. Treatment with Wnt3a, which elevates ß-catenin protein levels, also generated the increment of Smads proteins contents when comparing with untreated cells. In conclusion, IFN-α2b and TGF-ß1 proved to be effective as modulators of Wnt/ß-catenin pathway in HCC cell lines holding both wild-type and truncated ß-catenin. Since the inhibition of ß-catenin/TCF4/Smads complexes formation may have a critical role in slowing down oncogenesis, IFN-α2b and TGF-ß1 could be useful as potential treatments in patients with HCC.

TGF-beta receptors, in a Smad-independent manner, are required for terminal skeletal muscle differentiation.

Skeletal muscle differentiation is strongly inhibited by transforming growth factor type beta (TGF-beta), although muscle formation as well as regeneration normally occurs in an environment rich in this growth factor. In this study, we evaluated the role of intracellular regulatory Smads proteins as well as TGF-beta-receptors (TGF-beta-Rs) during skeletal muscle differentiation. We found a decrease of TGF-beta signaling during differentiation. This phenomenon is explained by a decline in the levels of the regulatory proteins Smad-2, -3, and -4, a decrease in the phosphorylation of Smad-2 and lost of nuclear translocation of Smad-3 and -4 in response to TGF-beta. No change in the levels and inhibitory function of Smad-7 was observed. In contrast, we found that TGF-beta-R type I (TGF-beta-RI) and type II (TGF-beta-RII) increased on the cell surface during skeletal muscle differentiation. To analyze the direct role of the serine/threonine kinase activities of TGF-beta-Rs, we used the specific inhibitor SB 431542 and the dominant-negative form of TGF-beta-RII lacking the cytoplasmic domain. The TGF-beta-Rs were important for successful muscle formation, determined by the induction of myogenin, creatine kinase activity, and myosin. Silencing of Smad-2/3 expression by specific siRNA treatments accelerated myogenin, myosin expression, and myotube formation; although when SB 431542 was present inhibition in myosin induction and myotube formation was observed, suggesting that these last steps of skeletal muscle differentiation require active TGF-beta-Rs. These results suggest that both down-regulation of Smad regulatory proteins and cell signaling through the TGF-beta receptors independent of Smad proteins are essential for skeletal muscle differentiation.

Molecular interaction of BMP-4, TGF-beta, and estrogens in lactotrophs: impact on the PRL promoter.

The regulatory role of estrogen, bone morphogenetic protein-4 (BMP-4), and TGF-beta has a strong impact on hormone secretion, gene transcription, and cellular growth of prolactin (PRL)-producing cells. In contrast to TGF-beta, BMP-4 induces the secretion of PRL in GH3 cells. Therefore, we studied the mechanism of their transcriptional regulation. Both BMP-4 and TGF-beta inhibited the transcriptional activity of the estrogen receptor (ER). Estrogens had no effect on TGF-beta-specific Smad protein transcriptional activity but presented a stimulatory action on the transcriptional activity of the BMP-4-specific Smads. BMP-4/estrogen cross talk was observed both on PRL hormone secretion and on the PRL promoter. This cross talk was abolished by the expression of a dominant-negative form for Smad-1 and treatment with ICI 182780 but not by point mutagenesis of the estrogen response element site within the promoter, suggesting that Smad/ER interaction might be dependent on the ER and a Smad binding element. By serial deletions of the PRL promoter, we observed that indeed a region responsive to BMP-4 is located between -2000 and -1500 bp upstream of the transcriptional start site. Chromatin immunoprecipitation confirmed Smad-4 binding to this region, and by specific mutation and gel shift assay, a Smad binding element responsible site was characterized. These results demonstrate that the different transcriptional factors involved in the Smad/ER complexes regulate their transcriptional activity in differential ways and may account for the different regulatory roles of BMP-4, TGF-beta, and estrogens in PRL-producing cells.