Hematopoietic stem cell-derived functional osteoblasts exhibit therapeutic efficacy in a murine model of osteogenesis imperfecta.

Currently, there is no cure for osteogenesis imperfecta (OI)-a debilitating pediatric skeletal dysplasia. Herein we show that hematopoietic stem cell (HSC) therapy holds promise in treating OI. Using single-cell HSC transplantation in lethally irradiated oim/oim mice, we demonstrate significant improvements in bone morphometric, mechanics, and turnover parameters. Importantly, we highlight that HSCs cause these improvements due to their unique property of differentiating into osteoblasts/osteocytes, depositing normal collagen-an attribute thus far assigned only to mesenchymal stem/stromal cells. To confirm HSC plasticity, lineage tracing was done by transplanting oim/oim with HSCs from two specific transgenic mice-VavR, in which all hematopoietic cells are GFP+ and pOBCol2.3GFP, where GFP is expressed only in osteoblasts/osteocytes. In both models, transplanted oim/oim mice demonstrated GFP+ HSC-derived osteoblasts/osteocytes in bones. These studies unequivocally establish that HSCs differentiate into osteoblasts/osteocytes, and HSC transplantation can provide a new translational approach for OI.

Hematopoietic stem cells are pluripotent and not just "hematopoietic".

Over a decade ago, several preclinical transplantation studies suggested the striking concept of the tissue-reconstituting ability (often referred to as HSC plasticity) of hematopoietic stem cells (HSCs). While this heralded an exciting time of radically new therapies for disorders of many organs and tissues, the concept was soon mired in controversy and remained dormant for almost a decade. This commentary provides a concise review of evidence for HSC plasticity, including more recent findings based on single HSC transplantation in mouse and clinical transplantation studies. There is strong evidence for the concept that HSCs are pluripotent and are the source for the majority, if not all, of the cell types in our body. Also discussed are some biological and experimental issues that need to be considered in the future investigation of HSC plasticity.

Hematopoietic stem cells give rise to osteo-chondrogenic cells.

Repair of bone fracture requires recruitment and proliferation of stem cells with the capacity to differentiate to functional osteoblasts. Given the close association of bone and bone marrow (BM), it has been suggested that BM may serve as a source of these progenitors. To test the ability of hematopoietic stem cells (HSCs) to give rise to osteo-chondrogenic cells, we used a single HSC transplantation paradigm in uninjured bone and in conjunction with a tibial fracture model. Mice were lethally irradiated and transplanted with a clonal population of cells derived from a single enhanced green fluorescent protein positive (eGFP+) HSC. Analysis of paraffin sections from these animals showed the presence of eGFP+ osteocytes and hypertrophic chondrocytes. To determine the contribution of HSC-derived cells to fracture repair, non-stabilized tibial fracture was created. Paraffin sections were examined at 7 days, 2 weeks and 2 months after fracture and eGFP+ hypertrophic chondrocytes, osteoblasts and osteocytes were identified at the callus site. These cells stained positive for Runx-2 or osteocalcin and also stained for eGFP demonstrating their origin from the HSC. Together, these findings strongly support the concept that HSCs generate bone cells and suggest therapeutic potentials of HSCs in fracture repair.

Quantitative increase in T regulatory cells enhances bone remodeling in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by repeated bone fractures. Recent studies have shown that T lymphocytes and regulatory T cells (Tregs) regulate the functions of osteoclasts and osteoblasts, thus playing a role in bone turnover. We demonstrate an activated effector phenotype and higher secretion of pro-inflammatory cytokines, IFN-γ, and TNF-α in OI peripheral T cells as compared with wild-type (WT). Suppressive Tregs (spleen and thymus) were qualitatively similar, whereas there was a quantitative decrease in OI versus WT. Restoring Treg numbers by systemic transplantation in OI mice resulted in reduced T cell activation and effector cytokine secretion that correlated with significant improvements in tibial trabecular and cortical bone parameters and stiffness of femur, along with increased osteoblast mineralization and decreased osteoclast numbers. Therefore, Tregs can dampen the pro-inflammatory environment and enhance bone remodeling in OI mice. Thus, this study will be helpful in developing future autologous immunotherapy-based treatment modalities for OI.

Carbon Monoxide Activates PERK-Regulated Autophagy to Induce Immunometabolic Reprogramming and Boost Antitumor T-cell Function.

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control. SIGNIFICANCE: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy.

MCP1 directs trafficking of hematopoietic stem cell-derived fibroblast precursors in solid tumor.

Our previous studies have demonstrated that hematopoietic stem cells (HSCs) are a novel source of carcinoma-associated fibroblasts. However, the mechanisms regulating recruitment and homing of HSC-derived carcinoma-associated fibroblasts or their precursors to the tumor microenvironment are unknown. Herein, we demonstrate using a single cell transplantation model that circulating fibroblast precursors (CFPs) are of HSC origin. This population increased with tumor burden in vivo and functional in vitro studies showed that CFPs preferentially migrated and differentiated into fibroblasts in response to tumor, suggesting that HSC-derived CFPs serve as an intermediate between the bone marrow and tumor. Based on this chemotactic ability and our demonstration of a monocyte lineage origin for CFPs, we investigated the role of monocyte chemoattractant protein (MCP1) in mediating CFP recruitment/homing. Blocking tumor-produced MCP1 inhibited in vitro migration of CFPs in response to multiple tumor types, indicating broad biological significance for this CFP/chemokine interaction. In vivo, CCR2-expressing CFPs increased in circulation during the period of active tumor growth and stromal development. Inhibition of MCP1 during tumor development resulted in decreased tumor volume in tumor-bearing mice. Together these findings confirm an HSC origin for CFPs, demonstrate a role for MCP1 in regulating their contribution to the tumor microenvironment, and suggest a potential therapeutic target for limiting tumor growth.

Targeting PIM Kinase with PD1 Inhibition Improves Immunotherapeutic Antitumor T-cell Response.

PURPOSE: Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo-engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor. EXPERIMENTAL DESIGN: The role of PIM kinases in T cells was studied either by genetic ablation (PIM1-/-PIM2-/-PIM3-/-) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutaneously and treated by transferring tumor epitope gp100-reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both. RESULTS: With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination. CONCLUSIONS: This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Pro-Survival Lipid Sphingosine-1-Phosphate Metabolically Programs T Cells to Limit Anti-tumor Activity.

Sphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.

Overexpression of COX-2 in human osteosarcoma cells decreases proliferation and increases apoptosis.

Overexpression of cyclooxygenase-2 (COX-2) is generally considered to promote tumorigenesis. To investigate a potential role of COX-2 in osteosarcoma, we overexpressed COX-2 in human osteosarcoma cells. Saos-2 cells deficient in COX-2 expression were retrovirally transduced or stably transfected with murine COX-2 cDNA. Functional expression of COX-2 was confirmed by Northern and Western analyses and prostaglandin production. Overexpression of COX-2 reduced cell numbers by 50% to 70% compared with controls. Decreased proliferation in COX-2-overexpressing cells was associated with cell cycle prolongation in G(2)-M. Apoptosis, measured by both Annexin V binding assay and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, was increased in cells overexpressing COX-2, and the increase was not reversed by treatment with NS-398, indicating that the effects were not mediated by prostaglandins. Retroviral COX-2 overexpression in two other human osteosarcoma cell lines, U2OS and TE85, also decreased cell viability. However, in the human colon carcinoma HCT-116 cell line, which is deficient in COX-2, retroviral overexpression of COX-2, at similar efficiency as in Saos-2 cells, increased resistance to apoptosis. Reactive oxygen species (ROS), measured by flow cytometry, were increased by COX-2 overexpression in Saos-2 cells but not in HCT-116 cells. Inhibition of peroxidase activity, but not of COX activity, blocked the ROS increase. Antioxidants blocked the increase in ROS and the increase in apoptosis due to COX-2 overexpression in Saos-2 cells. Our results suggest that (a) COX-2 overexpression in osteosarcoma cells may increase resistance to tumorigenesis by increasing ROS to levels that decrease cell viability and (b) the effects of COX-2 overexpression are cell type/tissue dependent.

Hematopoietic Stem Cells as a Novel Source of Dental Tissue Cells.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.

CD38-NAD+Axis Regulates Immunotherapeutic Anti-Tumor T Cell Response.

Heightened effector function and prolonged persistence, the key attributes of Th1 and Th17 cells, respectively, are key features of potent anti-tumor T cells. Here, we established ex vivo culture conditions to generate hybrid Th1/17 cells, which persisted long-term in vivo while maintaining their effector function. Using transcriptomics and metabolic profiling approaches, we showed that the enhanced anti-tumor property of Th1/17 cells was dependent on the increased NAD+-dependent activity of the histone deacetylase Sirt1. Pharmacological or genetic inhibition of Sirt1 activity impaired the anti-tumor potential of Th1/17 cells. Importantly, T cells with reduced surface expression of the NADase CD38 exhibited intrinsically higher NAD+, enhanced oxidative phosphorylation, higher glutaminolysis, and altered mitochondrial dynamics that vastly improved tumor control. Lastly, blocking CD38 expression improved tumor control even when using Th0 anti-tumor T cells. Thus, strategies targeting the CD38-NAD+ axis could increase the efficacy of anti-tumor adoptive T cell therapy.

Plasticity of hematopoietic stem cells.

Almost two decades ago, a number of cell culture and preclinical transplantation studies suggested the striking concept of the tissue-reconstituting ability of hematopoietic stem cells (HSCs). While this heralded an exciting time of radically new therapies for disorders of many organs and tissues, the concept was soon mired by controversy and remained dormant. This chapter provides a brief review of evidence for HSC plasticity including our findings based on single HSC transplantation in mouse. These studies strongly support the concept that HSCs are pluripotent and may be the source for the majority, if not all, of the cell types in our body.

Hematopoietic Origin of Murine Lung Fibroblasts.

Multiple origins, including the bone marrow, have been suggested to contribute to fibroblast populations in the lung. Using bone marrow reconstitution strategies, the present study tested the hypothesis that the bone marrow hematopoietic stem cell (HSC) gives rise to lung tissue fibroblasts in vivo. Data demonstrate that the nonadherent bone marrow fraction is enriched for CD45(+) HSC-derived cells and was able to reconstitute hematopoiesis in lethally irradiated animals. Analysis of peripheral blood and lung tissues from engrafted mice demonstrated the ability of this population to give rise to CD45(+)/Discoidin-Domain Receptor-2(+) (DDR2) circulating fibroblast precursors (CFPs) in blood and fibroblast populations in lung. An HSC origin for lung fibroblasts was confirmed using a novel clonal cell transplantation method in which the bone marrow is reconstituted by a clonal population derived from a single HSC. Together, these findings provide evidence for an HSC contribution to lung fibroblasts and demonstrate a circulating intermediate through the CD45(+)/DDR2(+) HSC-derived CFP.

Hematopoietic stem cell-derived adipocytes and fibroblasts in the tumor microenvironment.

The tumor microenvironment (TME) is complex and constantly evolving. This is due, in part, to the crosstalk between tumor cells and the multiple cell types that comprise the TME, which results in a heterogeneous population of tumor cells and TME cells. This review will focus on two stromal cell types, the cancer-associated adipocyte (CAA) and the cancer-associated fibroblast (CAF). In the clinic, the presence of CAAs and CAFs in the TME translates to poor prognosis in multiple tumor types. CAAs and CAFs have an activated phenotype and produce growth factors, inflammatory factors, cytokines, chemokines, extracellular matrix components, and proteases in an accelerated and aberrant fashion. Through this activated state, CAAs and CAFs remodel the TME, thereby driving all aspects of tumor progression, including tumor growth and survival, chemoresistance, tumor vascularization, tumor invasion, and tumor cell metastasis. Similarities in the tumor-promoting functions of CAAs and CAFs suggest that a multipronged therapeutic approach may be necessary to achieve maximal impact on disease. While CAAs and CAFs are thought to arise from tissues adjacent to the tumor, multiple alternative origins for CAAs and CAFs have recently been identified. Recent studies from our lab and others suggest that the hematopoietic stem cell, through the myeloid lineage, may serve as a progenitor for CAAs and CAFs. We hypothesize that the multiple origins of CAAs and CAFs may contribute to the heterogeneity seen in the TME. Thus, a better understanding of the origin of CAAs and CAFs, how this origin impacts their functions in the TME, and the temporal participation of uniquely originating TME cells may lead to novel or improved anti-tumor therapeutics.

Amelioration of a mouse model of osteogenesis imperfecta with hematopoietic stem cell transplantation: microcomputed tomography studies.

OBJECTIVE: To test the hypothesis that hematopoietic stem cells (HSCs) generate bone cells using bone marrow (BM) cell transplantation in a mouse model of osteogenesis imperfecta (OI). OI is a genetic disorder resulting from abnormal amount and/or structure of type I collagen and is characterized by osteopenia, fragile bones, and skeletal deformities. Homozygous OI murine mice (oim; B6C3Fe a/a-Col1a2(oim)/J) offer excellent recipients for transplantation of normal HSCs, because fast turnover of osteoprogenitors has been shown. MATERIALS AND METHODS: We transplanted BM mononuclear cells or 50 BM cells highly enriched for HSCs from transgenic enhanced green fluorescent protein mice into irradiated oim mice and analyzed changes in bone parameters using longitudinal microcomputed tomography. RESULTS: Dramatic improvements were observed in three-dimensional microcomputed tomography images of these bones 3 to 6 months post-transplantation when the mice showed high levels of hematopoietic engraftment. Histomorphometric assessment of the bone parameters, such as trabecular structure and cortical width, supported observations from three-dimensional images. There was an increase in bone volume, trabecular number, and trabecular thickness with a concomitant decrease in trabecular spacing. Analysis of a nonengrafted mouse or a mouse that was transplanted with BM cells from oim mice showed continued deterioration in the bone parameters. The engrafted mice gained weight and became less prone to spontaneous fractures while the control mice worsened clinically and eventually developed kyphosis. CONCLUSIONS: These findings strongly support the concept that HSCs generate bone cells. Furthermore, they are consistent with observations from clinical transplantation studies and suggest therapeutic potentials of HSCs in OI.

Hematopoietic stem cell origin of human fibroblasts: cell culture studies of female recipients of gender-mismatched stem cell transplantation and patients with chronic myelogenous leukemia.

OBJECTIVE: Our series of studies using transplantation of single hematopoietic stem cells (HSCs) demonstrated that mouse fibroblasts/myofibroblasts are derived from HSCs. In order to determine the origin of human fibroblasts, we established a method for culturing fibroblasts from human peripheral blood (PB) mononuclear cells and studied fibroblasts from gender-mismatched HSC transplant recipients and patients with untreated Philadelphia chromosome-positive chronic myelogenous leukemia (CML). MATERIALS AND METHODS: We cultured PB cells from three female subjects who showed near-complete hematopoietic reconstitution from transplantation of granulocyte-colony stimulating factor-mobilized male PB cells and examined the resulting fibroblasts using fluorescent in situ hybridization for Y chromosome. Because the mobilized PB cells may contain mesenchymal stem cells, we could not determine the HSC or mesenchymal stem cell origin of the fibroblasts seen in culture. To further document the HSC origin of human fibroblasts, we next examined fibroblasts from two patients with untreated CML, a known clonal disorder of HSCs. RESULTS: All cultured fibroblasts from female recipients of male cells showed the presence of Y chromosome, indicating the donor origin of fibroblasts. Cultured fibroblasts from the CML patients revealed the presence of BCR-ABL translocation. This demonstration provided strong evidence for the HSC origin of human fibroblasts because CML is a clonal disorder of the HSC. CONCLUSIONS: These studies strongly suggest that human fibroblasts are derived from HSCs. In addition, the results suggest that fibrosis seen in patients with CML may be a part of the clonal process.

Hematopoietic stem cell origin of adipocytes.

OBJECTIVE: It has generally been believed that adipocytes are derived from mesenchymal stem cells via fibroblasts. We recently reported that fibroblasts/myofibroblasts in a number of tissues and organs are derived from hematopoietic stem cells (HSCs). In the present study, we tested the hypothesis that HSCs also give rise to adipocytes. MATERIALS AND METHODS: Using transplantation of a single enhanced green fluorescent protein-positive (EGFP(+)) HSC and primary culture, we examined generation of adipocytes from HSCs. RESULTS: Adipose tissues from clonally engrafted mice showed EGFP(+) adipocytes that stained positive for leptin, perilipin, and fatty acid binding protein 4. A diet containing rosiglitazone, a peroxisome proliferator-activated receptor-gamma agonist, significantly enhanced the number of EGFP(+) adipocytes. When EGFP(+) bone marrow cells from clonally engrafted mice were cultured under adipogenic conditions, all of the cultured cells stained positive with Oil Red O and Sudan Black B and exhibited the presence of abundant mRNA for adipocyte markers. Finally, clonal culture- and sorting-based studies of Mac-1 expression of hematopoietic progenitors suggested that adipocytes are derived from HSCs via progenitors for monocytes/macrophages. CONCLUSION: Together, these studies clarify the current controversy regarding the ability of HSCs to give rise to adipocytes. Furthermore, our primary culture method that generates adipocytes from uncommitted hematopoietic cells should contribute to the studies of the mechanisms of early adipocytic differentiation and may lead to development of therapeutic solutions for many general obesity issues.

Inhibition of superoxide generation upon T-cell receptor engagement rescues Mart-1(27-35)-reactive T cells from activation-induced cell death.

Cytotoxic T lymphocytes (CTL) may undergo massive expansion upon appropriate antigenic stimulation. Homeostasis is maintained by a subsequent "contraction" of these cells. Activation-induced cell death (AICD) and programmed cell death prevent the untoward side effects, arising from excessive numbers and prolonged persistence of activated CTL, that occur upon uncontrolled and/or continued expansion. However, effector cell persistence has been identified as a hallmark of successful T-cell-mediated adoptive immunotherapy. Thus, prevention of AICD may be critical to achieve more successful clinical results. We have previously shown that treatment with the c-Jun NH(2)-terminal kinase (JNK) inhibitor SP600125 protects human melanoma epitope Mart-1(27-35)-reactive CTL from apoptotic death upon their reencounter with cognate antigen. However, inhibition of JNK also interferes with the functional ability of the CTL to secrete IFN-gamma. Here, we show that reactive oxygen species (ROS) inhibitors, such as the superoxide dismutase mimetic Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), efficiently protected Mart-1(27-35)-reactive primary CTL from AICD without impairing their functional capability. MnTBAP prevented the increase in intracellular ROS, mitochondrial membrane collapse, and DNA fragmentation observed in control-treated cells upon cognate antigen encounter. Furthermore, the mechanism of AICD prevention in primary CTL included blockade of JNK activation. Finally, tumor-reactive in vitro expanded tumor infiltrating lymphocytes, which are used clinically in cancer immunotherapy, also benefit from MnTBAP-mediated antioxidant treatment. Thus, modulation of the redox pathway might improve CTL persistence and lead to better clinical results for T cell-based immunotherapies.

Role of Cbfa1/Runx2 in the fluid shear stress induction of COX-2 in osteoblasts.

Induction of cyclooxygenase-2 (COX-2) is thought to be important for the anabolic effects of mechanical loading. The transcription factor Cbfa1/Runx2 is essential for osteoblastic differentiation. We examined the role of Cbfa1 in the fluid shear stress (FSS) induction of COX-2 in MC3T3-E1 cells stably transfected with a COX-2 promoter-luciferase reporter. Cells were subjected to FSS for 30 min and returned to static culture (post-FSS). COX-2 mRNA and promoter activity peaked 0.5-1h and 2-3h, respectively, post-FSS. Mutation of the Cbfa1 consensus sequence at -267/-261 bp decreased the FSS fold-induction of luciferase activity by 50%. On electrophoretic mobility shift assay (EMSA), proteins binding to an oligonucleotide spanning the Cbfa1 site were supershifted by specific antibody to Cbfa1. FSS did not increase Cbfa1 binding on EMSA or Cbfa1 mRNA or protein levels. These data suggest that transcriptional activity of Cbfa1, independent of its level of expression, is necessary for maximal FSS induction of COX-2 in osteoblasts.

Fluid flow induces Rankl expression in primary murine calvarial osteoblasts.

Mechanical loading of bone generates fluid flow within the mineralized matrix that exerts fluid shear stress (FSS) on cells. We examined effects of FSS on receptor activator of nuclear factor kappa B ligand (RANKL), a critical factor for osteoclast formation. Primary murine osteoblasts were subjected to pulsatile FSS (5 Hz, 10 dynes/cm(2)) for 1 h and then returned to static culture for varying times (post-FSS). Protein levels were measured by Western analysis and mRNA by Northern analysis, RT-PCR and quantitative PCR. There were 20- to 40-fold increases in RANKL mRNA at 2-4 h post-FSS. RANKL protein was induced by 2 h post-FSS and remained elevated for at least 8 h. Effects were independent of cyclooxygenase-2 activity. Small increases (up to three-fold) in mRNA of the decoy receptor for RANKL, osteoprotegerin, were seen. Five min of FSS, followed by static culture, was as effective in stimulating RANKL mRNA as 4 h of continuous FSS. FSS induced cAMP activity, and H-89, a protein kinase A (PKA) inhibitor, blocked the FSS induction of RANKL. H-89 also inhibited the PKC pathway, but specific PKC inhibitors, GF109203X and Go6983, did not inhibit FSS-induced RANKL. FSS induced phosphorylation of ERK1/2, and PD98059, an inhibitor of the ERK pathway, inhibited the FSS induction of RANKL mRNA 60%-90%. Thus, brief exposure to FSS resulted in sustained induction of RANKL expression after stopping FSS, and this induction was dependent on PKA and ERK signaling pathways. Increased RANKL after mechanical loading may play a role in initiating bone remodeling.