KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration.

Upon transplantation, skeletal stem cells (also known as bone marrow stromal or mesenchymal stem cells) can regulate bone regeneration by producing secreted factors. Here, we identify KIAA1199 as a bone marrow stromal cell-secreted factor in vitro and in vivo. KIAA1199 plasma levels of patients positively correlate with osteoporotic fracture risk and expression levels of KIAA1199 in patient bone marrow stromal cells negatively correlates with their osteogenic differentiation potential. KIAA1199-deficient bone marrow stromal cells exhibit enhanced osteoblast differentiation in vitro and ectopic bone formation in vivo. Consistently, KIAA1199 knockout mice display increased bone mass and biomechanical strength, as well as an increased bone formation rate. They also exhibit accelerated healing of surgically generated bone defects and are protected from ovariectomy-induced bone loss. Mechanistically, KIAA1199 regulates osteogenesis by inhibiting the production of osteopontin by osteoblasts, via integrin-mediated AKT and ERK-MAPK intracellular signaling. Thus, KIAA1199 is a regulator of osteoblast differentiation and bone regeneration and could be targeted for the treatment or management of low bone mass conditions.

Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation.

BACKGROUND: Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. METHODS: We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. RESULTS: In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. CONCLUSION: hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC.

KIAA1199 is a secreted molecule that enhances osteoblastic stem cell migration and recruitment.

Factors mediating mobilization of osteoblastic stem and progenitor cells from their bone marrow niche to be recruited to bone formation sites during bone remodeling are poorly known. We have studied secreted factors present in the bone marrow microenvironment and identified KIAA1199 (also known as CEMIP, cell migration inducing hyaluronan binding protein) in human bone biopsies as highly expressed in osteoprogenitor reversal cells (Rv.C) recruited to the eroded surfaces (ES), which are the future bone formation sites. In vitro, KIAA1199 did not affect the proliferation of human osteoblastic stem cells (also known as human bone marrow skeletal or stromal stem cells, hMSCs); but it enhanced cell migration as determined by scratch assay and trans-well migration assay. KIAA1199 deficient hMSCs (KIAA1199down) exhibited significant changes in cell size, cell length, ratio of cell width to length and cell roundness, together with reduction of polymerization actin (F-actin) and changes in phos-CFL1 (cofflin1), phos-LIMK1 (LIM domain kinase 1) and DSTN (destrin), key factors regulating actin cytoskeletal dynamics and cell motility. Moreover, KIAA1199down hMSC exhibited impaired Wnt signaling in TCF-reporter assay and decreased expression of Wnt target genes and these effects were rescued by KIAA1199 treatment. Finally, KIAA1199 regulated the activation of P38 kinase and its associated changes in Wnt-signaling. Thus, KIAA1199 is a mobilizing factor that interacts with P38 and Wnt signaling, and induces changes in actin cytoskeleton, as a mechanism mediating recruitment of hMSC to bone formation sites.

Actin depolymerization enhances adipogenic differentiation in human stromal stem cells.

Human stromal stem cells (hMSCs) differentiate into adipocytes that play a role in skeletal tissue homeostasis and whole body energy metabolism. During adipocyte differentiation, hMSCs exhibit significant changes in cell morphology suggesting changes in cytoskeletal organization. Here, we examined the effect of direct modulation of actin microfilament dynamics on adipocyte differentiation. Stabilizing actin filaments in hMSCs by siRNA-mediated knock down of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) or treating the cells by Phalloidin reduced adipocyte differentiation as evidenced by decreased number of mature adipocytes and decreased adipocyte specific gene expression (ADIPOQ, LPL, PPARG, FABP4). In contrast, disruption of actin cytoskeleton by Cytochalasin D enhanced adipocyte differentiation. Follow up studies revealed that the effects of CFL1 on adipocyte differentiation depended on the activity of LIM domain kinase 1 (LIMK1) which is the major upstream kinase of CFL1. Inhibiting LIMK by its specific chemical inhibitor LIMKi inhibited the phosphorylation of CFL1 and actin polymerization, and enhanced the adipocyte differentiation. Moreover, treating hMSCs by Cytochalasin D inhibited ERK and Smad2 signaling and this was associated with enhanced adipocyte differentiation. On the other hand, Phalloidin enhanced ERK and Smad2 signaling, but inhibited adipocyte differentiation which was rescued by ERK specific chemical inhibitor U0126. Our data provide a link between restructuring of hMSCs cytoskeleton and hMSCs lineage commitment and differentiation.

Osterix acetylation at K307 and K312 enhances its transcriptional activity and is required for osteoblast differentiation.

Osterix (Osx) is an essential transcription factor involved in osteoblast differentiation and bone formation. The precise molecular mechanisms of the regulation of Osx expression are not fully understood. In the present study, we found that in cells, both endogenous and exogenous Osx protein increased after treatment with histone deacetylase inhibitors Trichostatin A and hydroxamic acid. Meanwhile, the results of immunoprecipitation indicated that Osx was an acetylated protein and that the CREB binding protein (CBP), and less efficiently p300, acetylated Osx. The interaction and colocalization of CBP and Osx were demonstrated by Co-immunoprecipitation and immunofluorescence, respectively. In addition, K307 and K312 were identified as the acetylated sites of Osx. By contrast, HDAC4, a histone deacetylase (HDAC), was observed to interact and co-localize with Osx. HDAC4 was demonstrated to mediate the deacetylation of Osx. Moreover, we found that acetylation of Osx enhanced its stability, DNA binding ability and transcriptional activity. Finally, we demonstrated that acetylation of Osx was required for the osteogenic differentiation of C2C12 cells. Taken together, our results provide evidence that CBP-mediated acetylation and HDAC4-mediated deacetylation have critical roles in the modification of Osx, and thus are important in osteoblast differentiation.

Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells.

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover, depolymerizing actin reduced FAK, p38 and JNK activation during OB differentiation of hMSCs, while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation.

Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis.

Recent studies have revealed that osthole, an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson, a traditional Chinese medicine, possesses anticancer activity. However, its effect on breast cancer cells so far has not been elucidated clearly. In the present study, we evaluated the effects of osthole on the proliferation, cell cycle and apoptosis of human breast cancer cells MDA-MB 435. We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells, The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole, as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation. The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression. Were observed taken together, these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

Phosphorylation of Serine422 increases the stability and transactivation activities of human Osterix.

Osterix (Osx) is an essential regulator for osteoblast differentiation and bone formation. Although phosphorylation has been reported to be involved in the regulation of Osx activity, the precise underlying mechanisms remain to be elucidated. Here we identified S422 as a novel phosphorylation site of Osx and demonstrated that GSK-3ß interacted and co-localized with Osx. GSK-3ß increased the stability and transactivation activity of Osx through phosphorylation of the newly identified site. These findings expanded our understanding of the mechanisms of posttranslational regulation of Osx and the role of GSK-3ß in the control of Osx transactivation activity.

MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells.

Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified miRNA-34a (miR-34a) and its target protein networks as modulator of osteoblastic (OB) differentiation of hMSC. miRNA array profiling and further validation by quantitative RT-PCR revealed that miR-34a was upregulated during OB differentiation of hMSC, and in situ hybridization confirmed its OB expression in vivo. Overexpression of miR-34a inhibited early commitment and late OB differentiation of hMSC in vitro, whereas inhibition of miR-34a by anti-miR-34a enhanced these processes. Target prediction analysis and experimental validation confirmed Jagged1 (JAG1), a ligand for Notch 1, as a bona fide target of miR-34a. siRNA-mediated reduction of JAG1 expression inhibited OB differentiation. Moreover, a number of known cell cycle regulator and cell proliferation proteins, such as cyclin D1, cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), E2F transcription factor three, and cell division cycle 25 homolog A were among miR-34a targets. Furthermore, in a preclinical model of in vivo bone formation, overexpression of miR-34a in hMSC reduced heterotopic bone formation by 60%, and conversely, in vivo bone formation was increased by 200% in miR-34a-deficient hMSC. miRNA-34a exhibited unique dual regulatory effects controlling both hMSC proliferation and OB differentiation. Tissue-specific inhibition of miR-34a might be a potential novel therapeutic strategy for enhancing in vivo bone formation.

MicroRNA-214 suppresses osteogenic differentiation of C2C12 myoblast cells by targeting Osterix.

Osterix (Osx) is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. Osx-null mice, which exhibit a complete absence of bone formation and arrested osteoblast differentiation, die immediately after birth. However, our understanding of the regulatory mechanism of Osx expression remains poor. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play pivotal roles in diverse biological processes, including the development, differentiation, proliferation, survival, and oncogenesis of cells and organisms. In this study, we aimed to investigate the impact of miRNAs on Osx expression. Bioinformatic analyses predicted that miR-214 would be a potential regulator of Osx. The direct binding of miR-214 to the Osx 3' untranslated region (3' UTR) was demonstrated by a luciferase reporter assay using a construct containing the Osx 3' UTR. Deletion mutant construction revealed that the Osx 3' UTR contained two miR-214 binding sites. MiR-214 expression was inversely correlated with Osx expression in Saos-2 and U2OS cells. The forced expression of miR-214 in Saos-2 cells led to a reduction in the level of Osx protein. Moreover, the role of miR-214 in the osteogenic differentiation of C2C12 cells was investigated. We found that the osteogenic differentiation of C2C12 cells was enhanced by the downregulation of miR-214 expression, as measured by increased alkaline phosphatase activity and matrix mineralization. Taken together, these results indicate that miR-214 is a novel regulator of Osx, and that it plays an important role in the osteogenic differentiation of C2C12 cells as a suppressor.

Characterization of Osterix protein stability and physiological role in osteoblast differentiation.

Osterix (Osx/SP7) is a C2H2 zinc finger-containing transcription factor of the SP gene family. Osx knockout mice indicate that the gene plays an essential role in osteoblast differentiation and bone formation. However, the mechanisms involved in the regulation of Osx are still poorly understood. Here, we report a novel post-translational mechanism for the regulation of Osx in mammalian cells. We found that the stability of endogenous and exogenous Osx reduced after cycloheximide treatment. In cells treated with the proteasome inhibitors MG-132 or lactacystin, both endogenous and exogenous Osx protein expression increased in a time-dependent manner. Co-immunoprecipitation (Co-IP) assays showed that both endogenous and exogenous Osx were ubiquitinated. Six lysine residues of Osx were identified as candidate ubiquitination sites by construction of point mutant plasmids and luciferase reporter assays. Furthermore, we confirmed that K58 and K230 are the ubiquitination sites of Osx by Co-IP assays and protein stability assays. Moreover, the Osx K58R and K230R mutations promoted the expression of osteoblast differentiation markers (alkaline phosphatase, collagen I and osteocalcin) and enhanced osteogenic differentiation in C2C12 cells. Taken together, our data indicate that Osx is an unstable protein, and that the ubiquitin-proteasome pathway is involved in the regulation of Osx and thereby regulates osteoblast differentiation.