Abhishek Chandra

Epidermal Growth Factor Receptor (EGFR) Signaling Promotes Proliferation and Survival in Osteoprogenitors by Increasing Early Growth Response 2 (EGR2) Expression

Background: Maintaining bone architecture requires continuous generation of osteoblasts from osteoprogenitors. Results: EGFR signaling stimulates the expression of transcription factor EGR2 to promote osteoprogenitor proliferation and survival. Conclusion: EGFR-induced EGR2 expression is critical for osteoprogenitor maintenance and new bone formation. Significance: Understanding the mechanism of growth factor regulation of osteoprogenitor pool is crucial for designing a new anabolic strategy to treat bone-related diseases. Maintaining bone architecture requires continuous generation of osteoblasts from osteoprogenitor pools. Our previous study of mice with epidermal growth factor receptor (EGFR) specifically inactivated in osteoblast lineage cells revealed that EGFR stimulates bone formation by expanding the population of mesenchymal progenitors. EGFR ligands are potent regulators for the osteoprogenitor pool, but the underlying mechanisms are largely unknown. Here we demonstrate that activation of EGFR increases the number of osteoprogenitors by promoting cell proliferation and suppressing either serum depletion-induced or TNFα-induced apoptosis mainly through the MAPK/ERK pathway. Mouse calvarial organ culture revealed that EGF elevated the number of proliferative cells and decreased the number of apoptotic cells, which led to increased osteoblasts. Microarray analysis of MC3T3 cells, an osteoprogenitor cell line, revealed that EGFR signaling stimulates the expression of MCL1, an antiapoptotic protein, and a family of EGR transcription factors (EGR1, -2, and -3). The up-regulation of MCL1 and EGR2 by EGF was further confirmed in osteoprogenitors close to the calvarial bone surface. Overexpression of NAB2, a co-repressor for EGRs, attenuated the EGF-induced increase in osteoprogenitor number. Interestingly, knocking down the expression of EGR2, but not EGR1 or -3, resulted in a similar effect. Using inhibitor, adenovirus overexpression, and siRNA approaches, we demonstrate that EGFR signaling activates the MAPK/ERK pathway to stimulate the expression of EGR2, which in turn leads to cell growth and MCL1-mediated cell survival. Taken together, our data clearly demonstrate that EGFR-induced EGR2 expression is critical for osteoprogenitor maintenance and new bone formation.

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.

PTH1–34 alleviates radiotherapy-induced local bone loss by improving osteoblast and osteocyte survival

Cancer radiotherapy is often complicated by a spectrum of changes in the neighboring bone from mild osteopenia to osteoradionecrosis. We previously reported that parathyroid hormone (PTH, 1-34), an anabolic agent for osteoporosis, reversed bone structural deterioration caused by multiple microcomputed tomography (microCT) scans in adolescent rats. To simulate clinical radiotherapy for cancer patients and to search for remedies, we focally irradiated the tibial metaphyseal region of adult rats with a newly available small animal radiation research platform (SARRP) and treated these rats with intermittent injections of PTH1-34. Using a unique 3D image registration method that we recently developed, we traced the local changes of the same trabecular bone before and after treatments, and observed that, while radiation caused a loss of small trabecular elements leading to significant decreases in bone mass and strength, PTH1-34 preserved all trabecular elements in irradiated bone with remarkable increases in bone mass and strength. Histomorphometry demonstrated that SARRP radiation severely reduced osteoblast number and activity, which were impressively reversed by PTH treatment. In contrast, suppressing bone resorption by alendronate failed to rescue radiation-induced bone loss and to block the rescue effect of PTH1-34. Furthermore, histological analyses revealed that PTH1-34 protected osteoblasts and osteocytes from radiation-induced apoptosis and attenuated radiation-induced bone marrow adiposity. Taken together, our data strongly support a robust radioprotective effect of PTH on trabecular bone integrity through preserving bone formation and shed light on further investigations of an anabolic therapy for radiation-induced bone damage.

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.

Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH- Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTHs anabolic actions on bone.

3D image registration is critical to ensure accurate detection of longitudinal changes in trabecular bone density, microstructure, and stiffness measurements in rat tibiae by in vivo microcomputed tomography (μCT).

In the recent decade, in vivo μCT scanners have become available to monitor temporal changes in rodent bone in response to diseases and treatments. We investigated short-term and long-term precision of in vivo μCT measurements of trabecular bone density, microstructure and stiffness of rat tibiae and tested whether they can be improved by 3D image registration. Rats in the short-term precision group underwent baseline and follow-up scans within the same day (n = 15) and those in the long-term precision group were scanned at day 0 and day 14 (n = 16) at 10.5 μm voxel size. A 3D image-registration scheme was applied to register the trabecular bone compartments of baseline and follow-up scans. Prior to image registration, short-term precision ranged between 0.85% and 2.65% in bone volume fraction (BV/TV), trabecular number, thickness, and spacing (Tb.N*, Tb.Th*, Tb.Sp*), trabecular bone mineral density and tissue mineral density (Tb.BMD, and Tb.TMD), and was particularly high in structure model index (SMI), connectivity density (Conn.D), and stiffness (4.29%-8.83%). Image registration tended to improve the short-term precision, but the only statistically significant improvement was in Tb.N*, Tb.TMD, and stiffness. On the other hand, unregistered comparisons between day-0 and day-14 scans suggested significant increases in BV/TV, Tb.N*, Tb.Th*, Conn.D, and Tb.BMD and decrease in Tb.Sp* and SMI. However, the percent change in each parameter from registered comparisons was significantly different from unregistered comparisons. Registered results suggested a significant increase in BV/TV, Tb.BMD, and stiffness over 14 days, primarily caused by increased Tb.Th* and Tb.TMD. Due to the continuous growth of rodents, the direct comparisons between the unregistered baseline and follow-up scans were driven by changes due to global bone modeling instead of local remodeling. Our results suggested that 3D image registration is critical for detecting changes due to bone remodeling activities in rodent trabecular bone by in vivo μCT imaging.

Suppression of Sclerostin Alleviates Radiation-Induced Bone Loss by Protecting Bone-Forming Cells and Their Progenitors Through Distinct Mechanisms

Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/β‐catenin pathway can overcome radiation‐induced DNA damage and apoptosis of osteoblastic cells. Neutralization of circulating sclerostin with a monoclonal antibody (Scl‐Ab) is an innovative approach for treating osteoporosis by enhancing Wnt/β‐catenin signaling in bone. Together with the fact that focal radiation increases sclerostin amount in bone, we sought to determine whether weekly treatment with Scl‐Ab would prevent focal radiotherapy‐induced osteoporosis in mice. Micro‐CT and histomorphometric analyses demonstrated that Scl‐Ab blocked trabecular bone structural deterioration after radiation by partially preserving osteoblast number and activity. Consistently, trabecular bone in sclerostin null mice was resistant to radiation via the same mechanism. Scl‐Ab accelerated DNA repair in osteoblasts after radiation by reducing the number of γ‐H2AX foci, a DNA double‐strand break marker, and increasing the amount of Ku70, a DNA repair protein, thus protecting osteoblasts from radiation‐induced apoptosis. In osteocytes, apart from using similar DNA repair mechanism to rescue osteocyte apoptosis, Scl‐Ab restored the osteocyte canaliculi structure that was otherwise damaged by radiation. Using a lineage tracing approach that labels all mesenchymal lineage cells in the endosteal bone marrow, we demonstrated that radiation damage to mesenchymal progenitors mainly involves shifting their fate to adipocytes and arresting their proliferation ability but not inducing apoptosis, which are different mechanisms from radiation damage to mature bone forming cells. Scl‐Ab treatment partially blocked the lineage shift but had no effect on the loss of proliferation potential. Taken together, our studies provide proof‐of‐principle evidence for a novel use of Scl‐Ab as a therapeutic treatment for radiation‐induced osteoporosis and establish molecular and cellular mechanisms that support such treatment.

Quantification of skeletal growth, modeling, and remodeling by in vivo micro computed tomography

In this study we established an image analysis scheme for the investigation of cortical and trabecular bone development during skeletal growth and tested this concept on in vivo μCT images of rats. To evaluate its efficacy, we applied the technique to young (1-month-old) and adult (3-month-old) rat tibiae with vehicle (Veh) or intermittent parathyroid hormone (PTH) treatment. By overlaying 2 sequential scans based on their distinct trabecular microarchitecture, we calculated the linear growth rate of young rats to be 0.31 mm/day at the proximal tibia. Due to rapid growth (3.7 mm in 12 days), the scanned bone region at day 12 had no overlap with the bone tissue scanned at day 0. Instead, the imaged bone region at day 12 represented newly generated bone tissue from the growth plate. The new bone of the PTH-treated rats had significantly greater trabecular bone volume fraction, number, and thickness than those of the Veh-treated rats, indicating PTHs anabolic effect on bone modeling. In contrast, the effect of PTH on adult rat trabecular bone was found to be caused by PTHs anabolic effect on bone remodeling. The cortical bone at the proximal tibia of young rats also thickened more in the PTH group (23%) than the Veh group (14%). This was primarily driven by endosteal bone formation and coalescence of trabecular bone into the cortex. This process can be visualized by aligning the local bone structural changes using image registration. As a result, the cortex after PTH treatment was 31% less porous, and had a 22% greater polar moment of inertia compared to the Veh group. Lastly, we monitored the longitudinal bone growth in adult rats by measuring the distance of bone flow away from the proximal tibial growth plate from 3 months to 19 months of age and discovered a total of 3.5mm growth in 16 months. It was demonstrated that this image analysis scheme can efficiently evaluate bone growth, bone modeling, and bone remodeling, and is ready to be translated into a clinical imaging platform.

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.