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Human periodontal ligament stem cells (hPDLSCs) are mesenchymal stem cells (MSCs) derived from dental and craniofacial tissues that exhibit high potential for differentiation into osteoblasts. Recently, microRNAs (miRNAs) have been established to play important roles in MSC osteogenesis. In the current study, we report that miR-21 was down-regulated in osteogenically differentiated PDLSCs. Overexpression of miR-21 significantly inhibited osteogenesis of hPDLSC, whereas its inhibition demonstrated the opposite effects. Furthermore, SMAD family member 5 (Smad5) was predicted to be a downstream target of miR-21 and was shown to undergo up-regulation in PDLSCs induced toward osteogenesis. Moreover, Smad5 and Runx2, which are the critical transcription factors in osteogenic differentiation, were predicted to be targets of miR-21. Suppression of miR-21 expression increased the level of Smad5 in vitro and during in vivo transplantation experiments. Furthermore, suppression of Smad5 inhibited osteogenic differentiation and decreased the protein level of Runx2. Taken together, these results suggested that miR-21 be mechanistically implicated in the regulation of osteogenic differentiation of hPDLSCs by targeting Smad5.

Occlusal force is an important stimulus for maintaining periodontal homeostasis. This is attributed to the quality of human periodontal ligament fibroblasts (hPDLFs) that could transfer occlusal force into biological signals modulating osteoblst differentiation. However, few studies investigated the mechanism of occlusal force-induced osteodifferentiation of hPDLFs. In our study, we used the cyclic mechanical tension (CMT) at 10% elongation with 0.5 Hz to mimic occlusal force, and explored its effects on osteogenesis of hPDLFs. Firstly, elevated expressions of several osteoblast marker genes (Runx2, ATF4, SP7, OCN, and BSP), as well as activated ERK1/2 pathway were detected during CMT loading for 1, 3, 6, 12, 18, and 24 h. To gain further insight into how CMT contributed to those effects, we focused on the classic ERK1/2-Runx2 pathway by inhibiting ERK1/2 and overexpressing Runx2. Our results reflected that Runx2 overexpression alone could induce osteodifferentiation of hPDLFs. Meanwhile, CMT loading could intensify while combined ERK1/2 blockage could weaken this process. Furthermore, we found that CMT promoted Runx2 transcription and phosphorylation via ERK1/2; protein level of phospho-Runx2 (p-Runx2), rather than Runx2, was in parallel with mRNA expressions of SP7, OCN, and BSP. Taken together, our study proved that p-Runx2, elevated by CMT via ERK1/2 pathway, is the predominate factor in promoting osteoblast differentiation of hPDLFs.

BackgroundInterstitial pneumonia in connective tissue diseases (CTD-IP) featuring inflammation and fibrosis is a leading cause of death in CTD-IP patients. The related autoimmune lung injury and disturbed self-healing process make conventional anti-inflammatory drugs ineffective. Equipped with unique immunoregulatory and regenerative properties, mesenchymal stem cells (MSCs) may represent a promising therapeutic agent in CTD-IP. In this study, we aim to define the immunopathology involved in pulmonary exacerbation during autoimmunity and to determine the potential of MSCs in correcting these disorders.MethodsLung and blood specimens, bronchoalveolar lavage fluid cells collected from CTD-IP patients, and human primary lung fibroblasts (HLFs) from patients pathologically diagnosed with usual interstitial pneumonia (UIP) and healthy controls were analyzed by histology, flow cytometry and molecular biology. T cell subsets involved in the process of CTD-IP were defined, while the regulatory functions of MSCs isolated from the bone marrow of normal individuals (HBMSCs) on cytotoxic T cells and CTD-UIP HLFs were investigated in vitro.ResultsHigher frequencies of cytotoxic T cells were observed in the lung and peripheral blood of CTD-IP patients, accompanied with a reduced regulatory T cell (Treg) level. CTD-UIP HLFs secreted proinflammatory cytokines in combination with upregulation of α-smooth muscle actin (α-SMA). The addition of HBMSCs in vitro increased Tregs concomitant with reduced cytotoxic T cells in an experimental cell model with dominant cytotoxic T cells, and promoted Tregs expansion in T cell subsets from patients with idiopathic pulmonary fibrosis (IPF). HBMSCs also significantly decreased proinflammatory chemokine/cytokine expression, and blocked α-SMA activation in CTD-UIP HLFs through a TGF-β1-mediated mechanism, which modulates excessive IL-6/STAT3 signaling leading to IP-10 expression. MSCs secreting a higher level of TGF-β1 appear to have an optimal anti-fibrotic efficacy in BLM-induced pulmonary fibrosis in mice.ConclusionsImpairment of TGF-β signal transduction relevant to a persistent IL-6/STAT3 transcriptional activation contributes to reduction of Treg differentiation in CTD-IP and to myofibroblast differentiation in CTD-UIP HLFs. HBMSCs can sensitize TGF-β1 downstream signal transduction that regulates IL-6/STAT3 activation, thereby stimulating Treg expansion and facilitating anti-fibrotic IP-10 production. This may in turn block progression of lung fibrosis in autoimmunity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0319-y) contains supplementary material, which is available to authorized users.

Metastasis and drug resistance are major barriers for the treatment of non-small cell lung cancer (NSCLC). To explore new therapeutic options, we successfully encapsulated MicroRNA-34a (miR-34a), a potent endogenous tumor suppressor in NSCLC into S6 aptamer-conjugated dendrimer to form lung cancer-targeted gene delivery nanoparticles (PAM-Ap/pMiR-34a NPs). PAM-Ap/pMiR-34a NPs had a diameter of 100–200 nm and Zeta potential of ~30 mV at applied N/P ratio. The aptamer conjugation significantly improved cellular uptake as well as gene transfection efficiency of PAM-Ap/pMiR-34a NPs in cultured NSCLC cells. We showed that PAM-Ap/pMiR-34a NPs enhanced the regulation of targeted genes, BCL-2 and p53 in vitro. In addition, we revealed PAM-Ap/pMiR-34a NPs significantly inhibited cell growth, migration, invasion and induced apoptosis of lung cancer cells compared with non-targeted NPs. The method provided a novel therapeutic strategy for the experimental treatment of NSCLC.

The present study investigated the effect of folic acid supplementation on the Notch signaling pathway and cell proliferation in rat embryonic neural stem cells (NSCs). The NSCs were isolated from E14–16 rat brain and grown as neurospheres in serum-free suspension culture. Individual cultures were assigned to one of 3 treatment groups that differed according to the concentration of folic acid in the medium: Control (baseline folic acid concentration of 4 mg/l), low folic acid supplementation (4 mg/l above baseline, Folate-L) and high folic acid supplementation (40 mg/l above baseline, Folate-H). NSCs were identified by their expression of immunoreactive nestin and proliferating cells by incorporation of 5'bromo-2'deoxyuridine. Cell proliferation was also assessed by methyl thiazolyl tetrazolium assay. Notch signaling was analyzed by real-time PCR and western blot analyses of the expression of Notch1 and hairy and enhancer of split 5 (Hes5). Supplementation of NSCs with folic acid increased the mRNA and protein expression levels of Notch1 and Hes5. Folic acid supplementation also stimulated NSC proliferation dose-dependently. Embryonic NSCs respond to folic acid supplementation with increased Notch signaling and cell proliferation. This mechanism may mediate the effects of folic acid supplementation on neurogenesis in the embryonic nervous system.

Mechanically stretched skeletal muscle undergoes dramatic shifts in structure, mass, and function. In vitro tensile strain models have demonstrated that myogenic progenitor cells, including satellite cells and myoblasts, are highly mechanosensitive cells, and respond to mechanical strain in a wide variety of aspects. However, the experimental results from different researchers and laboratories are not always in support of each other. Moreover, some specific molecules or signaling pathways were reported to play distinct roles in stretched myogenic cells, according to the statements of different studies. The purpose of this review is to integrate the researches conducting in vitro culture of satellite cells or myoblasts and exploring their mechanoresponses using in vitro stretching apparatus. These responses will be categorized into several groups, such as activation, proliferation, myogenic differentiation, cellular damage or apoptosis, properties of plasma membrane, transdifferentiation, reorientation, etc. In addition, detailed experimental designs like culturing conditions and straining regimens will be displayed and compared, to interpret some contradictory statements in different studies. Furthermore, the currently known interconnections among some mechanosensitive pathways will be pictured to give a better understanding about the complex regulations of myogenic cell responses to mechanical stretch. Hopefully, by summarizing the published studies about mechanoresponses of myogenic progenitor cells, future directions, and perspectives would be made clearer to researchers in this field.

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.