Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat non-unions. The use of MSC to treat fractures is attractive as it would be implementing a reparative process that should be in place but occurs to be defective or protracted and MSC effects would be needed only for the repairing time that is relatively brief. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed-tomography and histological studies. Transplanting CMV-Cre-R26R-LacZ-MSC, we found that MSC engrafted within the callus endosteal niche. Using MSC from BMP-2-Lac-Z mice genetically modified using a bacterial artificial chromosome system to be β-gal reporters for BMP-2 expression, we found that MSC contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow to design novel MSC-based therapies to treat fractures.

Introduction: Mesenchymal stem cells (MSCs) can be isolated from adult bone marrow (BM), expanded, and differentiated into several cell types, including chondrocytes. The role of IGF-I in the chondrogenic potential of MSCs is poorly understood. TGF-␤ induces MSC chondrogenic differentiation, although its actions are not well defined. The aim of our study was to define the biological role of IGF-I on proliferation, chondrogenic condensation, apoptosis, and differentiation of MSCs into chondrocytes, alone or in combination with TGF-␤ and in the presence or absence of TGF-␤ signaling. Materials and Methods: Mononuclear adherent stem cells were isolated from mouse BM. Chondrogenic differentiation was induced by culturing high-density MSC pellets in serum-and insulin-free defined medium up to 7 days, with or without IGF-I and/or TGF-␤. We measured thymidine incorporation and stained 2-day-old pellets with TUNEL, cleaved caspase-3, peanut-agglutinin, and N-cadherin. Seven-day-old pellets were measured in size, stained for proteoglycan synthesis, and analyzed for the expression of collagen II and Sox-9 by quantitative real time PCR. We obtained MSCs from mice in which green fluorescent protein (GFP) was under the Collagen2 promoter and determined GFP expression by confocal microscopy. We conditionally inactivated the TGF-␤ type II receptor (T␤RII) in MSCs using a cre-lox system, generating T␤RII knockout MSCs (RIIKO-MSCs). Results and Conclusions: IGF-I modulated MSC chondrogenesis by stimulating proliferation, regulating cell apoptosis, and inducing expression of chondrocyte markers. IGF-I chondroinductive actions were equally potent to TGF-␤1, and the two growth factors had additive effects. Using RIIKO-MSCs, we showed that IGF-I chondrogenic actions are independent from the TGF-␤ signaling. We found that the extracellular signalrelated kinase 1/2 mitogen-activated protein kinase (Erk1/2 MAPK) pathway mediated the TGF-␤1 mitogenic response and in part the IGF-I proliferative action. Our data, by showing the role of IGF-I and TGF-␤1 in the critical steps of MSC chondrogenesis, provide critical information to optimize the therapeutic use of MSCs in cartilage disorders.

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor β (TGF-β) signaling in mice lacking the TGF-β type II receptor gene (Tgfbr2) in their limbs (Tgfbr2PRX-1KO). In Tgfbr2PRX-1KO mice, the loss of TGF-β responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2Prx1KO joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2–green fluorescent protein–β–GEO–bacterial artificial chromosome β-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2PRX-1KO mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-β receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2PRX-1KO growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-β signaling represents a means of entry to initiate the process.

Integrins regulate cell-cell and cell-matrix adhesion and thereby play critical roles in tumor progression and metastasis. Although work in preclinical models suggests that β 1 integrins may stimulate metastasis of a number of cancers, expression of the β 1 subunit alone has not been shown to be a useful prognostic indicator in human cancer patients. Here we have demonstrated that the α 2 β 1 integrin suppresses metastasis in a clinically relevant spontaneous mouse model of breast cancer. These data are consistent with previous studies indicating high expression of α 2 β 1 integrin in normal breast epithelium and loss of α 2 β 1 in poorly differentiated breast cancer. They are also consistent with our systematic analysis of microarray databases of human breast and prostate cancer, which revealed that decreased expression of the gene encoding α 2 integrin, but not genes encoding α 1 , α 3 , or β 1 integrin, was predictive of metastatic dissemination and decreased survival. The predictive value of α 2 expression persisted within both good-risk and poor-risk cohorts defined by estrogen receptor and lymph node status. Thus, the α 2 β 1 integrin functionally inhibits breast tumor metastasis, and α 2 expression may serve as an important biomarker of metastatic potential and patient survival.

DNA polymerase ␣-primase is known to be phosphorylated in human and yeast cells in a cell cycledependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase ␣-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2-and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase ␣-primase from human cells, synchronized and labeled in G 1 /S and in G 2 , revealed a cyclin E/cdk2-like pattern in G 1 /S and a cyclin A/cdk2-like pattern in G 2 . The slower-electrophoretic-mobility form of p68 was absent in human cells in G 1 /S and appeared as the cells entered G 2 /M. Consistent with this, the ability of DNA polymerase ␣-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G 1 /S, decreased as the cells completed S phase, and reached a minimum in G 2 /M. These results suggest that the replication activity of DNA polymerase ␣-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.DNA replication in eukaryotic cells takes place during a restricted period of the cell cycle, the S phase. The transition from G 1 into S phase in vertebrate cells is regulated by at least two cyclin-dependent kinases, cyclin E/cdk2 and cyclin A/cdk2 (reviewed in references 37 and 38). Cyclin E/cdk2 activity peaks in late G 1 (14, 26), while cyclin A/cdk2 activity appears later, with the onset of DNA synthesis (42,44,54). Microinjection of either an anti-cyclin A antibody, an antisense cyclin A expression plasmid (18,40,55,63), or an anti-cyclin E antibody (39) prevented the entry of cells into S phase, documenting the importance of these cyclins for the G 1 -to-S transition. Interestingly, microinjection of anti-cyclin A antibodies after S-phase entry appeared to have no effect on DNA synthesis or S-phase progression (40), despite evidence that cyclin A/cdk2 resides in replication foci (5, 6, 47). However, cyclin A/cdk2 activity rises throughout S phase, and cyclin A is required again for the S/G 2 transition (40). The requirement for cyclin E/cdk2 and cyclin A/cdk2 activities for entry into S phase implies that they are needed to modify protein substrates involved in initiation of DNA replication, but relatively little is known about how phosphorylation of physiological substrates triggers initiation of vert...

SUMMARY Despite its clinical significance, the mechanisms of joint morphogenesis are still elusive. Here, we show by combining laser-capture microdissection for RNA sampling with microarray analysis, that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by down-regulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in-vivo, ex-vivo and in-vitro approaches, we showed that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2Prx1KO), which lack joint development and fail chondrocyte hypertrophy, showed up-regulation of interzone-MCP-5. In-vivo and ex-vivo blockade of the sole MCP-5 receptor, CCR2, in Tgfbr2Prx1KO led to rescue of joint formation and growth plate maturation; while in control mice determined an acceleration of endochondral growth plate mineralization. Taken together, we characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.

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BACKGROUND: Procoagulant states, leading to activation of the coagulation protease thrombin, are common in cancer and portend a poor clinical outcome. Although procoagulant states in osteosarcoma patients have been described, studies exploring osteosarcoma cells' ability to directly contribute to procoagulant activity have not been reported. This study explores the hypothesis that osteosarcoma can regulate thrombin generation and proliferate in response to thrombin, and that attenuating thrombin generation with anticoagulants can slow tumor growth. METHODS: Pathologic analysis of osteosarcoma with adjacent venous thrombus was performed. In vitro proliferation assays, cell-based coagulant activity assays, and quantification of coagulation cofactor expression were performed on human and murine osteosarcoma cell lines with varying aggressiveness. The efficacy of low molecular weight heparin (LMWH) attenuation of tumor-dependent thrombin generation and growth in vitro and in vivo was determined. RESULTS: Venous thrombi adjacent to osteosarcoma were found to harbor tumor surrounded by fibrin expressing coagulation cofactors, a finding associated with poor clinical outcome. More aggressive osteosarcoma cell lines had greater surface expression of procoagulant factors and generated more thrombin than less aggressive cell lines and were found to proliferate in response to thrombin. Treatment with LMWH reduced in vitro osteosarcoma proliferation and procoagulant activity as well as tumor growth in vivo. CONCLUSIONS: These findings suggest that elements of the coagulation cascade may play a role in and represent a pharmaceutical target to disrupt osteosarcoma growth. They also have broader implications, as they suggest that, to be effective, dosing of anticoagulants must take into account an individual tumor's capacity to generate thrombin. Cancer 2012;118:2494