Regis J. O’Keefe

Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair

Preclinical and clinical studies suggest a possible role for cyclooxygenases in bone repair and create concerns about the use of nonsteroidal antiinflammatory drugs in patients with skeletal injury. We utilized wild-type, COX-1(-/-), and COX-2(-/-) mice to demonstrate that COX-2 plays an essential role in both endochondral and intramembranous bone formation during skeletal repair. The healing of stabilized tibia fractures was significantly delayed in COX-2(-/-) mice compared with COX-1(-/-) and wild-type controls. The histology was characterized by a persistence of undifferentiated mesenchyme and a marked reduction in osteoblastogenesis that resulted in a high incidence of fibrous nonunion in the COX-2(-/-) mice. Similarly, intramembranous bone formation on the calvaria was reduced 60% in COX-2(-/-) mice following in vivo injection of FGF-1 compared with either COX-1(-/-) or wild-type mice. To elucidate the mechanism involved in reduced bone formation, osteoblastogenesis was studied in bone marrow stromal cell cultures obtained from COX-2(-/-) and wild-type mice. Bone nodule formation was reduced 50% in COX-2(-/-) mice. The defect in osteogenesis was completely rescued by addition of prostaglandin E2 (PGE(2)) to the cultures. In the presence of bone morphogenetic protein (BMP-2), bone nodule formation was enhanced to a similar level above that observed with PGE(2) alone in both control and COX-2(-/-) cultures, indicating that BMPs complement COX-2 deficiency and are downstream of prostaglandins. Furthermore, we found that the defect in COX-2(-/-) cultures correlated with significantly reduced levels of cbfa1 and osterix, two genes necessary for bone formation. Addition of PGE(2) rescued this defect, while BMP-2 enhanced cbfa1 and osterix in both COX-2(-/-) and wild-type cultures. Finally, the effects of these agents were additive, indicating that COX-2 is involved in maximal induction of osteogenesis. These results provide a model whereby COX-2 regulates the induction of cbfa1 and osterix to mediate normal skeletal repair.

A Perspective: Engineering Periosteum for Structural Bone Graft Healing

Autograft is superior to both allograft and synthetic bone graft in repair of large structural bone defect largely due to the presence of multipotent mesenchymal stem cells in periosteum. Recent studies have provided further evidence that activation, expansion and differentiation of the donor periosteal progenitor cells are essential for the initiation of osteogenesis and angiogenesis of donor bone graft healing. The formation of donor cell-derived periosteal callus enables efficient host-dependent graft repair and remodeling at the later stage of healing. Removal of periosteum from bone autograft markedly impairs healing whereas engraftment of multipotent mesenchymal stem cells on bone allograft improves healing and graft incorporation. These studies provide rationale for fabrication of a biomimetic periosteum substitute that could fit bone of any size and shape for enhanced allograft healing and repair. The success of such an approach will depend on further understanding of the molecular signals that control inflammation, cellular recruitment as well as mesenchymal stem cell differentiation and expansion during the early phase of the repair process. It will also depend on multidisciplinary collaborations between biologists, material scientists and bioengineers to address issues of material selection and modification, biological and biomechanical parameters for functional evaluation of bone allograft healing.

Regulation of chondrogenesis and chondrocyte differentiation by stress

Chondrogenesis and endochondral ossification are the cartilage differentiation processes that lead to skeletal formation and growth in the developing vertebrate as well as skeletal repair in the adult. The exquisite regulation of these processes, both in normal development and in pathologic situations, is impacted by a number of different types of stress. These include normal stressors such as mechanical loading and hypoxia as well pathologic stressors such as injury and/or inflammation and environmental toxins. This article provides an overview of the processes of chondrogenesis and endochondral ossification and their control at the molecular level. A summary of the influence of the most well-understood normal and pathologic stressors on the differentiation program is also presented.

Nfκb: A Pivotal Transcription Factor in Prostate Cancer Metastasis to Bone

Prostate cancers frequently metastasize to bone and this accounts for substantial morbidity. We investigated the potential role of the transcription factor NFκB as a central regulator of prostate cancer metastasis using the prostate adenocarcinoma cell line, PC-3, in a series of in vitro studies. Wild type PC-3 cells (PC-3.WT) have high basal levels of NFκB signaling, otherwise absent in PC-3 cells stably expressing a mutant form of the inhibitory kappa B (IκB) protein alpha (PC-3.mIκB). Although PC-3.WT cells in co-culture with rat bone marrow cells enhance bone resorption, no increase was observed in co-cultures with PC-3.mIκB cells. Similarly, although PC-3.WT cells were invasive in a chicken chorioallantoic membrane extravasation model, PC-3.mIκB cells lose this capacity to invade. NFκB reciprocally regulated genes involved in cellular invasion, with upregulation of MMP-9 and downregulation of its inhibitor, TIMP-1 in PC-3.WT cells, whereas MMP-9 was downregulated and TIMP-1 was upregulated in PC-3.mIκB cells. Finally, high basal gene and protein expression of the osteoclast-activating cytokine IL-6, observed in PC-3.WT cells, was absent in PC-3.mIκB cells. These in vitro experiments suggest NFκB as an important target to prevent prostate cancer bone metastasis and provide a rationale for further study of this transcription factor in metastatic disease.

Teriparatide as a Chondroregenerative Therapy for Injury-Induced Osteoarthritis

Teriparatide is chondroprotective and chondroregenerative in a mouse model of injury-induced osteoarthritis of the knee. Extending the Service Life of Arthritic Joints Every year, millions of people with osteoarthritis are forced to scale back their physical activities hoping to alleviate pain and increase the longevity of their degenerating joints. The problem is serious: A decade from now, 25% or more of the U.S. population are predicted to suffer from osteoarthritis. The hallmark problem in osteoarthritis is the progressive and irreversible loss of cartilage. Ultimately, the only option is to surgically replace the lost cartilage with metal and plastic. But are there alternative strategies that could lead to cartilage replacement and reduce the cycle of pain and reduced quality of life? In this issue of Science Translational Medicine, Sampson and colleagues report that a naturally occurring hormone called parathyroid hormone (trade name Forteo), already approved by the Food and Drug Administration to build bone, can also boost the buildup of cartilage in a mouse model of injury-induced osteoarthritis. In this mouse model, injury to the meniscus and ligaments of the knee initiates a slow process of cartilage degeneration that is comparable to that seen in people suffering a similar injury. To approximate the clinical situation of treating someone with symptomatic osteoarthritis caused by a past trauma, the researchers administered parathyroid hormone to mice that were already osteoarthritic because of an injury to the medial meniscus and medial collateral ligament. Tissue and molecular analyses of the injured knee joints revealed that after 1 month of daily treatment with parathyroid hormone, the cartilage layer was 32% thicker than in injured mice that did not receive the hormone. In addition, the investigators noted an increase in production of matrix molecules by chondrocytes (the cells that produce cartilage), suppression of genes associated with inappropriate chondrocyte maturation, and a reduction in cartilage breakdown. The ability of parathyroid hormone to boost the addition of new cartilage matrix while blocking its degradation in osteoarthritic joints suggests that it may be a useful therapeutic for treating patients with osteoarthritis, a pervasive clinical condition with surgery as the only current solution. There is no disease-modifying therapy for osteoarthritis, a degenerative joint disease that is projected to afflict more than 67 million individuals in the United States alone by 2030. Because disease pathogenesis is associated with inappropriate articular chondrocyte maturation resembling that seen during normal endochondral ossification, pathways that govern the maturation of articular chondrocytes are candidate therapeutic targets. It is well established that parathyroid hormone (PTH) acting via the type 1 PTH receptor induces matrix synthesis and suppresses maturation of chondrocytes. We report that the PTH receptor is up-regulated in articular chondrocytes after meniscal injury and in osteoarthritis in humans and in a mouse model of injury-induced knee osteoarthritis. To test whether recombinant human PTH(1–34) (teriparatide) would inhibit aberrant chondrocyte maturation and associated articular cartilage degeneration, we administered systemic teriparatide (Forteo), a Food and Drug Administration–approved treatment for osteoporosis, either immediately after or 8 weeks after meniscal/ligamentous injury in mice. Knee joints were harvested at 4, 8, or 12 weeks after injury to examine the effects of teriparatide on cartilage degeneration and articular chondrocyte maturation. Microcomputed tomography revealed increased bone volume within joints from teriparatide-treated mice compared to saline-treated control animals. Immediate systemic administration of teriparatide increased proteoglycan content and inhibited articular cartilage degeneration, whereas delayed treatment beginning 8 weeks after injury induced a regenerative effect. The chondroprotective and chondroregenerative effects of teriparatide correlated with decreased expression of type X collagen, RUNX2 (runt-related transcription factor 2), matrix metalloproteinase 13, and the carboxyl-terminal aggrecan cleavage product NITEGE. These preclinical findings provide proof of concept that Forteo may be useful for decelerating cartilage degeneration and inducing matrix regeneration in patients with osteoarthritis.

Cartilage-specific RBPjκ-dependent and -independent Notch signals regulate cartilage and bone development

The Notch signaling pathway has emerged as an important regulator of endochondral bone formation. Although recent studies have examined the role of Notch in mesenchymal and chondro-osteo progenitor cell populations, there has yet to be a true examination of Notch signaling specifically within developing and committed chondrocytes, or a determination of whether cartilage and bone formation are regulated via RBPjκ-dependent or -independent Notch signaling mechanisms. To develop a complete understanding of Notch signaling during cartilage and bone development we generated and compared general Notch gain-of-function (Rosa-NICDf/+), RBPjκ-deficient (Rbpjκf/f), and RBPjκ-deficient Notch gain-of-function (Rosa-NICDf/+;Rbpjκf/f) conditional mutant mice, where activation or deletion of floxed alleles were specifically targeted to mesenchymal progenitors (Prx1Cre) or committed chondrocytes (inducible Col2CreERT2). These data demonstrate, for the first time, that Notch regulation of chondrocyte maturation is solely mediated via the RBPjκ-dependent pathway, and that the perichodrium or osteogenic lineage probably influences chondrocyte terminal maturation and turnover of the cartilage matrix. Our study further identifies the cartilage-specific RBPjκ-independent pathway as crucial for the proper regulation of chondrocyte proliferation, survival and columnar chondrocyte organization. Unexpectedly, the RBPjκ-independent Notch pathway was also identified as an important long-range cell non-autonomous regulator of perichondral bone formation and an important cartilage-derived signal required for coordinating chondrocyte and osteoblast differentiation during endochondral bone development. Finally, cartilage-specific RBPjκ-independent Notch signaling likely regulates Ihh responsiveness during cartilage and bone development.

NF-κB Regulates VCAM-1 Expression on Fibroblast-Like Synoviocytes

Expression of VCAM-1 on synovial fibroblasts is a clinical hallmark of rheumatoid arthritis. The interaction of VCAM-1 and its integrin receptor very late Ag-4 is believed to be critically involved in the recruitment and retention of immune cells in the inflamed joints. To study the regulation of VCAM-1 in synovial fibroblasts, fibroblast-like synoviocytes (FLS) were isolated from the knee joints of normal mice and passaged repeatedly to obtain a homogeneous cell population. We have found that VCAM-1 is constitutively expressed on mouse FLS (mFLS) and that its surface expression is further increased after exposure to TNF-α. Nuclear translocation of transcription factor NF-κB including P50/P50 homodimer and P65/P50 heterodimer was activated by TNF-α treatment. In mFLS stably expressing a dominant-negative mutant of the inhibitory protein I-κBα- (mI-κB), which does not undergo proteolytic degradation, NF-κB remains in the cytosol and its activation in response to TNF-α is abolished. VCAM-1 protein expression after TNF-α stimulation was blocked in cells expressing the mI-κB. This effect is likely due to the loss of NF-κB-mediated transcription of VCAM-1, because the 5-fold increase in mRNA levels in response to TNF-α is absent in the mutant cells. To confirm these findings, we transduced mFLS with an adenoviral vector containing the mI-κB transgene. VCAM-1 expression was also blocked by mI-κB in this system, whereas cells transduced with a control adenoviral vector remained responsive to TNF-α. These results indicate that NF-κB mediates TNF-α-induced VCAM-1 expression on mFLS.

Inhibition of the Wnt-β-catenin and Notch signaling pathways sensitizes osteosarcoma cells to chemotherapy

Osteosarcoma (OS) is one of the most common malignant bone tumors in early adolescence. Multi-drug chemotherapy has greatly increased the five year survival rate from 20% to 70%. However, the rate has been staggering for 30 years and the prognosis is particularly poor for patients with recurrence and metastasis. Our study aimed to investigate the role of Wnt-β-catenin, Notch and Hedgehog pathway in OS development because all these pathways are involved in skeletal development, tumorigenesis and chemoresistance. Our results showed that the major components in Wnt-β-catenin pathway, e.g. Wnt3a, β-catenin and Lef1, were consistently upregulated in human osteosarcoma cell line Saos2 cells compared to human fetal osteoblasts (hFOB), whereas the changes in the expression levels of Notch and Hh signaling molecules were not consistent. Knocking down β-catenin increased the Saos2 sensitivity to methotrexate (MTX) induced cell death. Consistently, the expression level of β-catenin protein correlated with the invasiveness of OS, as evidenced by more intensive β-catenin immunoreactivity in higher grade OS samples. Chemical inhibition of the Wnt-β-catenin signaling enhanced MTX mediated death of Saos2 cells. A synergistic effect with MTX was observed when both inhibitors for Wnt-β-catenin and Notch pathways were simultaneously used, while the addition of the Hh inhibitor did not further improve the efficacy. Our findings provide some novel insight to OS pathogenesis and lay a foundation for future application of Wnt-β-catenin and Notch inhibitors together with the currently used chemotherapeutic drugs to improve the outcome of OS treatment.

Gene Expression Analysis of the Pleiotropic Effects of TGF-β1 in an In Vitro Model of Flexor Tendon Healing

Flexor tendon injuries are among the most challenging problems for hand surgeons and tissue engineers alike. Not only do flexor tendon injuries heal with poor mechanical strength, they can also form debilitating adhesions that may permanently impair hand function. While TGF-β1 is a necessary factor for regaining tendon strength, it is associated with scar and adhesion formation in the flexor tendons and other tissues as well as fibrotic diseases. The pleiotropic effects of TGF-β1 on tendon cells and tissue have not been characterized in detail. The goal of the present study was to identify the targets through which the effects of TGF-β1 on tendon healing could be altered. To accomplish this, we treated flexor tendon tenocytes cultured in pinned collagen gels with 1, 10 or 100 ng/mL of TGF-β1 and measured gel contraction and gene expression using RT-PCR up to 48 hours after treatment. Specifically, we studied the effects of TGF-β1 on the expression of collagens, fibronectin, proteoglycans, MMPs, MMP inhibitors, and the neotendon transcription factors, Scleraxis and Mohawk. Area contraction of the gels was not dose-dependent with the TGF-β1 concentrations tested. We observed dose-dependent downregulation of MMP-16 (MT3-MMP) and decorin, and upregulation of biglycan, collagen V, collagen XII, PAI-1, Scleraxis, and Mohawk by TGF-β1. Inter-gene analyses were also performed to further characterize the expression of ECM and MMP genes in the tenocyte-seeded collagen gels. These analyses illustrate that TGF-β1 tilts the balance of gene expression in favor of ECM synthesis rather than the matrix-remodeling MMPs, a possible means by which TGF-β1 promotes adhesion formation.

β‐catenin, cartilage, and osteoarthritis

The early cellular events during the development of osteoarthritis (OA) are accelerated articular chondrocyte maturation and extracellular matrix degradation, which are usually seen in the weight‐bearing region of articular cartilage. The results of our recent studies from transgenic OA mouse models indicate that upregulation of β‐catenin signaling in articular chondrocytes is most likely responsible for the conversion of normal articular chondrocytes into maturing (arthritic) chondrocytes, which is associated with activation of chondrocyte maturational genes and matrix degradation. Conditional activation of the β‐catenin gene in articular chondrocytes leads to an OA‐like phenotype. Overexpression of Smurf2, an E3 ubiquitin ligase, also induces an OA‐like phenotype through upregulation of β‐catenin signaling. In addition, β‐catenin upregulation was also found in articular cartilage tissues in patients with OA. These findings indicate that β‐catenin plays a central role in articular cartilage function and that activation of β‐catenin signaling may represent a pathologic mechanism for OA development.