Xianrong Zhang

EGFR signaling suppresses osteoblast differentiation and inhibits expression of master osteoblastic transcription factors Runx2 and Osterix.

The epidermal growth factor receptor (EGFR) and its ligands regulate key processes of cell biology, such as proliferation, survival, differentiation, migration, and tumorigenesis. We previously showed that, EGFR signaling pathway is an important bone regulator and it primarily plays an anabolic role in bone metabolism. In this study, we demonstrated that EGF‐like ligands strongly inhibited osteoblast differentiation and mineralization in several lines of osteoblastic cells. Real‐time RT‐PCR and promoter reporter assays revealed that EGF‐like ligands suppressed the expression of both early and late bone marker genes at the transcriptional level in the differentiating osteoblasts via an EGFR‐dependent manner. This inhibitory effect of EGFR signaling was not dependent on its mitogenic activity. Furthermore, we demonstrated that EGFR signaling reduced the expression of two major osteoblastic transcription factors Runx2 (type II) and Osterix in osteoblast differentiating cells. EGFR‐induced decrease in Runx2 transcriptional activity was confirmed by Runx2 reporter and chromatin immunoprecipitation assays. EGFR signaling increased the protein amounts of transcription co‐repressors HDAC4 and 6 and over‐expression of HDAC4 decreased Runx2 amount in differentiating osteoblasts, implying that HDACs contribute to the down‐regulation of Runx2 by EGFR. Moreover, activation of EGFR in undifferentiated osteoprogenitors attenuated the expression of early bone markers and Osterix and decreased Runx2 protein amounts. Together with our previous data, that EGFR stimulates osteoprogenitor proliferation and that blocking EGFR activity in osteoblast lineage cells results in fewer osteoprogenitors and an osteopenic phenotype, we conclude that EGFR signaling is important for maintaining osteoprogenitor population at an undifferentiated stage. J. Cell. Biochem. 112: 1749–1760, 2011.

The Critical Role of the Epidermal Growth Factor Receptor in Endochondral Ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR‐specific small‐molecule inhibitor, gefitinib, into 1‐month‐old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro‐osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP‐9, ‐13, and ‐14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF‐α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage‐specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro‐osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.

Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow.

Long bone is an anatomically complicated tissue with trabecular-rich metaphyses at two ends and cortical-rich diaphysis at the center. The traditional flushing method isolates only mesenchymal progenitor cells from the central region of long bones and these cells are distant from the bone surface. We propose that mesenchymal progenitors residing in endosteal bone marrow that is close to the sites of bone formation, such as trabecular bone and endosteum, behave differently from those in the central bone marrow. In this report, we separately isolated endosteal bone marrow using a unique enzymatic digestion approach and demonstrated that it contained a much higher frequency of mesenchymal progenitors than the central bone marrow. Endosteal mesenchymal progenitors express common mesenchymal stem cell markers and are capable of multi-lineage differentiation. However, we found that mesenchymal progenitors isolated from different anatomical regions of the marrow did exhibit important functional differences. Compared with their central marrow counterparts, endosteal mesenchymal progenitors have superior proliferative ability with reduced expression of cell cycle inhibitors. They showed greater immunosuppressive activity in culture and in a mouse model of inflammatory bowel disease. Aging is a major contributing factor for trabecular bone loss. We found that old mice have a dramatically decreased number of endosteal mesenchymal progenitors compared with young mice. Parathyroid hormone (PTH) treatment potently stimulates bone formation. A single PTH injection greatly increased the number of endosteal mesenchymal progenitors, particularly those located at the metaphyseal bone, but had no effect on their central counterparts. In summary, endosteal mesenchymal progenitors are more metabolically active and relevant to physiological bone formation than central mesenchymal progenitors. Hence, they represent a biologically important target for future mesenchymal stem cell studies.

Epidermal Growth Factor Receptor Plays an Anabolic Role in Bone Metabolism In Vivo

While the epidermal growth factor receptor (EGFR)–mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro–computed tomography (µCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col‐Cre Egfrf/f), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant‐negative allele, Wa5, and generated Col‐Cre EgfrWa5/f mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony‐forming unit–fibroblast (CFU‐F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild‐type mice caused a significant reduction in trabecular bone volume. In contrast, EgfrDsk5/+ mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism.

PTH prevents the adverse effects of focal radiation on bone architecture in young rats.

Radiation therapy is a common treatment regimen for cancer patients. However, its adverse effects on the neighboring bone could lead to fractures with a great impact on quality of life. The underlying mechanism is still elusive and there is no preventive or curative solution for this bone loss. Parathyroid hormone (PTH) is a current therapy for osteoporosis that has potent anabolic effects on bone. In this study, we found that focal radiation from frequent scans of the right tibiae in 1-month-old rats by micro-computed tomography severely decreased trabecular bone mass and deteriorated bone structure. Interestingly, PTH daily injections remarkably improved trabecular bone in the radiated tibiae with increases in trabecular number, thickness, connectivity, structure model index and stiffness, and a decrease in trabecular separation. Histomorphometric analysis revealed that radiation mainly decreased the number of osteoblasts and impaired their mineralization activity but had little effects on osteoclasts. PTH reversed these adverse effects and greatly increased bone formation to a similar level in both radiated and non-radiated bones. Furthermore, PTH protects bone marrow mesenchymal stem cells from radiation-induced damage, including a decrease in number and an increase in adipogenic differentiation. While radiation generated the same amount of free radicals in the bone marrow of vehicle-treated and PTH-treated animals, the percentage of apoptotic bone marrow cells was significantly attenuated in the PTH group. Taken together, our data demonstrate a radioprotective effect of PTH on bone structure and bone marrow and shed new light on a possible clinical application of anabolic treatment in radiotherapy.

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways

Background: EGFR is an important player in endochondral ossification. Results: Mice with EGFR deficiency in chondrocytes have delayed secondary ossification center formation due to reduced cartilage degradation, and EGFR regulates MMPs and RANKL expression in chondrocytes partially via Wnt/β-catenin. Conclusion: EGFR regulates epiphyseal cartilage development. Significance: The cross-talk between EGFR and Wnt/β-catenin signaling in chondrocytes shed new light on studying cartilage development and diseases. The epidermal growth factor receptor (EGFR) is an essential player in the development of multiple organs during embryonic and postnatal stages. To understand its role in epiphyseal cartilage development, we generated transgenic mice with conditionally inactivated EGFR in chondrocytes. Postnatally, these mice exhibited a normal initiation of cartilage canals at the perichondrium, but the excavation of these canals into the cartilage was strongly suppressed, resulting in a delay in the formation of the secondary ossification center (SOC). This delay was accompanied by normal chondrocyte hypertrophy but decreased mineralization and apoptosis of hypertrophic chondrocytes and reduced osteoclast number at the border of marrow space. Immunohistochemical analyses demonstrated that inactivation of chondrocyte-specific EGFR signaling reduced the amounts of matrix metalloproteinases (MMP9, -13, and -14) and RANKL (receptor activator of NF-κB ligand) in the hypertrophic chondrocytes close to the marrow space and decreased the cartilage matrix degradation in the SOC. Analyses of EGFR downstream signaling pathways in primary epiphyseal chondrocytes revealed that up-regulation of MMP9 and RANKL by EGFR signaling was partially mediated by the canonical Wnt/β-catenin pathway, whereas EGFR-enhanced MMP13 expression was not. Further biochemical studies suggested that EGFR signaling stimulates the phosphorylation of LRP6, increases active β-catenin level, and induces its nuclear translocation. In line with these in vitro studies, deficiency in chondrocyte-specific EGFR activity reduced β-catenin amount in hypertrophic chondrocytes in vivo. In conclusion, our work demonstrates that chondrocyte-specific EGFR signaling is an important regulator of cartilage matrix degradation during SOC formation and epiphyseal cartilage development and that its actions are partially mediated by activating the β-catenin pathway.

Reduced EGFR signaling enhances cartilage destruction in a mouse osteoarthritis model.

Osteoarthritis (OA) is a degenerative joint disease and a major cause of pain and disability in older adults. We have previously identified epidermal growth factor receptor (EGFR) signaling as an important regulator of cartilage matrix degradation during epiphyseal cartilage development. To study its function in OA progression, we performed surgical destabilization of the medial meniscus (DMM) to induce OA in two mouse models with reduced EGFR activity, one with genetic modification (EgfrWa5/+ mice) and the other one with pharmacological inhibition (gefitinib treatment). Histological analyses and scoring at 3 months post-surgery revealed increased cartilage destruction and accelerated OA progression in both mouse models. TUNEL staining demonstrated that EGFR signaling protects chondrocytes from OA-induced apoptosis, which was further confirmed in primary chondrocyte culture. Immunohistochemistry showed increased aggrecan degradation in these mouse models, which coincides with elevated amounts of ADAMTS5 and matrix metalloproteinase 13 (MMP13), the principle proteinases responsible for aggrecan degradation, in the articular cartilage after DMM surgery. Furthermore, hypoxia-inducible factor 2α (HIF2α), a critical catabolic transcription factor stimulating MMP13 expression during OA, was also upregulated in mice with reduced EGFR signaling. Taken together, our findings demonstrate a primarily protective role of EGFR during OA progression by regulating chondrocyte survival and cartilage degradation.

Loss of β-catenin induces multifocal periosteal chondroma-like masses in mice.

Osteochondromas and enchondromas are the most common tumors affecting the skeleton. Osteochondromas can occur as multiple lesions, such as those in patients with hereditary multiple exostoses. Unexpectedly, while studying the role of β-catenin in cartilage development, we found that its conditional deletion induces ectopic chondroma-like cartilage formation in mice. Postnatal ablation of β-catenin in cartilage induced lateral outgrowth of the growth plate within 2 weeks after ablation. The chondroma-like masses were present in the flanking periosteum by 5 weeks and persisted for more than 6 months after β-catenin ablation. These long-lasting ectopic masses rarely contained apoptotic cells. In good correlation, transplants of β-catenin-deficient chondrocytes into athymic mice persisted for a longer period of time and resisted replacement by bone compared to control wild-type chondrocytes. In contrast, a β-catenin signaling stimulator increased cell death in control chondrocytes. Immunohistochemical analysis revealed that the amount of detectable β-catenin in cartilage cells of osteochondromas obtained from hereditary multiple exostoses patients was much lower than that in hypertrophic chondrocytes in normal human growth plates. The findings in our study indicate that loss of β-catenin expression in chondrocytes induces periosteal chondroma-like masses and may be linked to, and cause, the persistence of cartilage caps in osteochondromas.

EGFR signaling is critical for maintaining the superficial layer of articular cartilage and preventing osteoarthritis initiation

Significance The uppermost superficial zone of articular cartilage plays multifaceted roles in maintaining cartilage structure, function, and mechanical properties and in preventing cartilage degeneration during osteoarthritis initiation. However, its regulation by growth factors and hormones is still largely unknown. Here we report that EGFR signaling is an important growth factor pathway that maintains superficial chondrocyte number, promotes boundary lubricant secretion and cartilage surface lubrication, and stimulates mechanical strength of articular cartilage. Reduction in EGFR activity leads to structurally, functionally, and mechanically compromised articular cartilage during development and drastically accelerates cartilage degeneration under normal and surgically induced osteoarthritis conditions. Thus, our studies strongly suggest that targeting cartilage surface EGFR signaling should be considered as a novel direction for osteoarthritis treatment. Osteoarthritis (OA) is the most common joint disease, characterized by progressive destruction of the articular cartilage. The surface of joint cartilage is the first defensive and affected site of OA, but our knowledge of genesis and homeostasis of this superficial zone is scarce. EGFR signaling is important for tissue homeostasis. Immunostaining revealed that its activity is mostly dominant in the superficial layer of healthy cartilage but greatly diminished when OA initiates. To evaluate the role of EGFR signaling in the articular cartilage, we studied a cartilage-specific Egfr-deficient (CKO) mouse model (Col2-Cre EgfrWa5/flox). These mice developed early cartilage degeneration at 6 mo of age. By 2 mo of age, although their gross cartilage morphology appears normal, CKO mice had a drastically reduced number of superficial chondrocytes and decreased lubricant secretion at the surface. Using superficial chondrocyte and cartilage explant cultures, we demonstrated that EGFR signaling is critical for maintaining the number and properties of superficial chondrocytes, promoting chondrogenic proteoglycan 4 (Prg4) expression, and stimulating the lubrication function of the cartilage surface. In addition, EGFR deficiency greatly disorganized collagen fibrils in articular cartilage and strikingly reduced cartilage surface modulus. After surgical induction of OA at 3 mo of age, CKO mice quickly developed the most severe OA phenotype, including a complete loss of cartilage, extremely high surface modulus, subchondral bone plate thickening, and elevated joint pain. Taken together, our studies establish EGFR signaling as an important regulator of the superficial layer during articular cartilage development and OA initiation.

Nanoindentation modulus of murine cartilage: a sensitive indicator of the initiation and progression of post-traumatic osteoarthritis

OBJECTIVE This study aims to demonstrate that cartilage nanoindentation modulus is a highly sensitive indicator of the onset and spatiotemporal progression of post-traumatic osteoarthritis (PTOA) in murine models. DESIGN Destabilization of the medial meniscus (DMM) surgery was performed on the right knees of 12-week old male, wild-type C57BL/6 mice, with Sham control on contralateral left knees. Atomic force microscopy (AFM)-based nanoindentation was applied to quantify the nanoindentation modulus, Eind, of femoral condyle cartilage at 3 days to 12 weeks after surgery. The modulus changes were compared against the timeline of histological OA signs. Meanwhile, at 8 weeks after surgery, changes in meniscus, synovium and subchondral bone were evaluated to reveal the spatial progression of PTOA. RESULTS The modulus of medial condyle cartilage was significantly reduced at 1 week after DMM, preceding the histological OA signs, which only became detectable at 4-8 weeks after. This reduction is likely due to concomitantly elevated proteolytic activities, as blocking enzymatic activities in mice can attenuate this modulus reduction. In later OA, lateral condyle cartilage and medial meniscus also started to be weakened, illustrating the whole-organ nature of PTOA. CONCLUSIONS This study underscores the high sensitivity of nanoindentation in examining the initiation, attenuation and progression of PTOA in murine models. Meanwhile, modulus changes highlight concomitant changes in lateral cartilage and meniscus during the advancement of OA.