Molecular and functional mapping of Plekhm1-Rab7 interaction in osteoclasts.

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases.

Targeted Overexpression of Claudin 11 in Osteoblasts Increases Trabecular Bone Mass by Stimulating Osteogenesis at the Expense of Adipogenesis in Mice.

Mice lacking Claudin11 (Cldn11) manifest reduced trabecular bone mass. However, the impact of Cldn11 expression in osteoblasts in vivo remains understudied. Herein, we generated osteoblast-specific transgenic (Tg) mice expressing Cldn11 and characterized their skeletal phenotype. Micro-CT analyses of the distal metaphysis of the femur showed a 50% and a 38% increase in trabecular bone mass in Tg male and female mice, respectively, due to a significant increase in trabecular number and a reduction in trabecular separation. Histomorphometry and serum biomarker studies uncovered that increased trabecular bone mass in Cldn11 Tg mice was the consequence of enhanced bone formation. Accordingly, an abundance of bone formation (Alp, Bsp), but not bone resorption (Ctsk), markers were augmented in the femurs of Cldn11 Tg mice. Since the trabecular bone density is known to inversely correlate with the amount of marrow adipose tissue (MAT), we measured the MAT in osmium-tetroxide-labeled bones by micro-CT scanning. We found 86% less MAT in the proximal tibia of the Tg males. Consistently, the expression levels of the adipogenic markers, adiponectin and leptin, were 50% lower in the femurs of the Tg males. Our data are consistent with the possibility that claudin11 exerts anabolic effects in osteoblastic lineage cells that act via promoting the differentiation of marrow stem cells towards osteoblasts at the expense of adipocytes.

Global and Conditional Disruption of the Igf-I Gene in Osteoblasts and/or Chondrocytes Unveils Epiphyseal and Metaphyseal Bone-Specific Effects of IGF-I in Bone.

To evaluate the relative importance of IGF-I expression in various cell types for endochondral ossification, we quantified the trabecular bone at the secondary spongiosa and epiphysis of the distal femur in 8-12-week-old male mice with a global knockout of the Igf-I gene, as well as the conditional deletion of Igf-I in osteoblasts, chondrocytes, and osteoblasts/chondrocytes and their corresponding wild-type control littermates. The osteoblast-, chondrocyte-, and osteoblast/chondrocyte-specific Igf-I conditional knockout mice were generated by crossing Igf-I floxed mice with Cre transgenic mice in which Cre expression is under the control of either the Col1α2 or Col2α1 promoter. We found that the global disruption of Igf-I resulted in 80% and 70% reductions in bone size, defined as total volume, at the secondary spongiosa and epiphysis of the distal femur, respectively. The abrogation of Igf-I in Col1α2-producing osteoblasts but not Col2α1-producing chondrocytes decreased bone size by 25% at both the secondary spongiosa and epiphysis. In comparison, the deletion of the Igf-I globally or specifically in osteoblasts or chondrocytes reduced trabecular bone mass by 25%. In contrast, the universal deletion of Igf-I in all cells, but not the conditional disruption of Igf-I in osteoblasts and/or chondrocytes reduced trabecular bone mass in the epiphysis. The reduced trabecular bone mass at the secondary spongiosa in osteoblast- and/or chondrocyte-specific Igf-I conditional knockout mice is caused by the reduced trabecular number and increased trabecular separation. Immunohistochemistry studies found that the expression levels of chondrocyte (COL10, MMP13) and osteoblast (BSP) markers were less in the secondary spongiosa and the epiphyses in the global Igf-I deletion mice. Our data indicate that local and endocrine Igf-I act pleiotropically and in a cell type- and bone compartment-dependent manner in bone.

WNT16 Regulation of the Articular Chondrocyte Phenotype in Mice.

The anabolic effects of WNT16 on osteoblasts are well established, however, little is known regarding the role of WNT16 in chondrocytes. In this study, we evaluated Wnt16 expression and its biological effects on mouse articular chondrocytes (ACs), since these cells are key to the development of osteoarthritis. While ACs derived from the long bone epiphysis of 7-day old C57BL/6J mice express multiple Wnts, Wnt5b and Wnt16 represent the two most highly expressed Wnts (expressed at several-fold higher levels than other Wnts). Treatment of serum-free AC cultures, with 100 ng/mL of recombinant human (rh) WNT16 for 24 h (hrs), increased proliferation (20%, p < 0.05) and expression levels of makers (Sox9 and Col2) of immature chondrocytes at both 24 h and 72 h, while Acan increased at 72 h. Expression of Mmp9, a marker of mature chondrocytes was decreased at 24 h. Additionally, WNT16 treatment regulated expression levels of Wnt ligands in a biphasic manner, inhibiting its expression at 24 h, while stimulating expression at 72 h. To determine whether WNT16 exerted anabolic effects on the AC phenotype, ex vivo cultures of tibial epiphyses were treated with rhWNT16 or vehicle for 9 days, and the articular cartilage phenotype was evaluated by safranin O cartilage staining and expression of articular cartilage marker genes. Both articular cartilage area and expression levels of AC markers were increased after rhWNT16 treatment. Our data suggest that Wnt16 expressed in ACs may play a role in regulating joint cartilage homeostasis via its direct effect, as well as through modulating the expression of other Wnt ligands.

Leucine Repeat Rich Kinase 1 Controls Osteoclast Activity by Managing Lysosomal Trafficking and Secretion.

We previously demonstrated that mice with targeted deletion of the leucine repeat rich kinase 1 (Lrrk1) gene were osteopetrotic due to the failure of osteoclasts to resorb bone. To determine how LRRK1 regulates osteoclast activity, we examined the intracellular and extracellular acidification with an acidotropic probe, acridine orange, in live osteoclasts on bone slices. We examined lysosome distribution in osteoclasts by localization of LAMP-2, cathepsin K, and v-ATPase by immunofluorescent staining with specific antibodies. We found that both vertical and horizontal cross-sectional images of the wild-type (WT) osteoclasts showed orange-staining of the intracellular acidic vacuoles/lysosomes dispersed to the ruffled border. By contrast, the LRRK1 deficient osteoclasts exhibited fluorescent orange staining in the cytoplasm away from the extracellular lacunae because of an altered distribution of the acidic vacuoles/lysosomes. In addition, WT osteoclasts displayed a peripheral distribution of LAMP-2 positive lysosomes with a typical actin ring. The clustered F-actin constitutes a peripheral sealing zone and a ruffled border which was stretched out into a resorption pit. The LAMP-2 positive lysosomes were also distributed to the sealing zone, and the cell was associated with a resorption pit. By contrast, LRRK1-deficient osteoclasts showed diffused F-actin throughout the cytoplasm. The sealing zone was weak and not associated with a resorption pit. LAMP-2 positive lysosomes were also diffuse in the cytoplasm and were not distributed to the ruffled border. Although the LRRK1-deficient osteoclast expressed normal levels of cathepsin K and v-ATPase, the lysosomal-associated cathepsin K and v-ATPase were not accumulated at the ruffled border in Lrrk1 KO osteoclasts. Our data indicate that LRRK1 controls osteoclast activity by regulating lysosomal distribution, acid secretion, and protease exocytosis.

Development of an Animal Model for Traumatic Brain Injury Augmentation of Heterotopic Ossification in Response to Local Injury.

Heterotopic ossification (HO) is the abnormal growth of bone in soft connective tissues that occurs as a frequent complication in individuals with traumatic brain injury (TBI) and in rare genetic disorders. Therefore, understanding the mechanisms behind ectopic bone formation in response to TBI is likely to have a significant impact on identification of novel therapeutic targets for HO treatment. In this study, we induced repetitive mild TBI (mTBI) using a weight drop model in mice and then stimulated HO formation via a local injury to the Achilles tendon or fibula. The amount of ectopic bone, as evaluated by micro-CT analyses, was increased by four-fold in the injured leg of mTBI mice compared to control mice. However, there was no evidence of HO formation in the uninjured leg of mTBI mice. Since tissue injury leads to the activation of hypoxia signaling, which is known to promote endochondral ossification, we evaluated the effect of IOX2, a chemical inhibitor of PHD2 and a known inducer of hypoxia signaling on HO development in response to fibular injury. IOX2 treatment increased HO volume by five-fold compared to vehicle. Since pericytes located in the endothelium of microvascular capillaries are known to function as multipotent tissue-resident progenitors, we determined if activation of hypoxia signaling promotes pericyte recruitment at the injury site. We found that markers of pericytes, NG2 and PDGFRß, were abundantly expressed at the site of injury in IOX2 treated mice. Treatment of pericytes with IOX2 for 72 h stimulated expression of targets of hypoxia signaling (Vegf and Epo), as well as markers of chondrocyte differentiation (Col2α1 and Col10α1). Furthermore, serum collected from TBI mice was more effective in promoting the proliferation and differentiation of pericytes than control mouse serum. In conclusion, our data show that the hypoxic state at the injury site in soft tissues of TBI mice provides an environment leading to increased accumulation and activation of pericytes to form endochondral bone.

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair.

The increasing incidence of physiologic/pathologic conditions that impair the otherwise routine healing of endochondral bone fractures and the occurrence of severe bone injuries necessitate novel approaches to enhance clinically challenging bone fracture repair. To promote the healing of nonunion fractures, we tested an approach that used two small molecules to sequentially enhance cartilage development and conversion to the bone in the callus of a murine femoral segmental defect nonunion model of bone injury. Systemic injections of smoothened agonist 21k (SAG21k) were used to stimulate chondrogenesis through the activation of the sonic hedgehog (SHH) pathway early in bone repair, while injections of the prolyl hydroxylase domain (PHD)2 inhibitor, IOX2, were used to stimulate hypoxia signaling-mediated endochondral bone formation. The expression of SHH pathway genes and Phd2 target genes was increased in chondrocyte cell lines in response to SAG21k and IOX2 treatment, respectively. The segmental defect responded to sequential systemic administration of these small molecules with increased chondrocyte expression of PTCH1, GLI1, and SOX9 in response to SAG and increased expression of hypoxia-induced factor-1α and vascular endothelial growth factor-A in the defect tissues in response to IOX2. At 6 weeks postsurgery, the combined SAG-IOX2 therapy produced increased bone formation in the defect with the bony union over the injury. Clinical significance: This therapeutic approach was successful in promoting cartilage and bone formation within a critical-size segmental defect and established the utility of a sequential small molecule therapy for the enhancement of fracture callus development in clinically challenging bone injuries.

Contrasting effects of Ksr2, an obesity gene, on trabecular bone volume and bone marrow adiposity.

Pathological obesity and its complications are associated with an increased propensity for bone fractures. Humans with certain genetic polymorphisms at the kinase suppressor of ras2 (KSR2) locus develop severe early-onset obesity and type 2 diabetes. Both conditions are phenocopied in mice with Ksr2 deleted, but whether this affects bone health remains unknown. Here we studied the bones of global Ksr2 null mice and found that Ksr2 negatively regulates femoral, but not vertebral, bone mass in two genetic backgrounds, while the paralogous gene, Ksr1, was dispensable for bone homeostasis. Mechanistically, KSR2 regulates bone formation by influencing adipocyte differentiation at the expense of osteoblasts in the bone marrow. Compared with Ksr2's known role as a regulator of feeding by its function in the hypothalamus, pair-feeding and osteoblast-specific conditional deletion of Ksr2 reveals that Ksr2 can regulate bone formation autonomously. Despite the gains in appendicular bone mass observed in the absence of Ksr2, bone strength, as well as fracture healing response, remains compromised in these mice. This study highlights the interrelationship between adiposity and bone health and provides mechanistic insights into how Ksr2, an adiposity and diabetic gene, regulates bone metabolism.

Our bones are living tissues which constantly reshape and renew themselves. This ability relies on stem cells present in the marrow cavity, which can mature into the various types of cells needed to produce new bone material, marrow fat, or other components. Obesity and associated conditions such as type 2 diabetes are often linked to harmful changes in the skeleton. In particular, these metabolic conditions are associated with weight-bearing bones becoming more prone to facture and healing poorly. Mice genetically modified to model obesity and diabetes could help researchers to study exactly how these conditions ­ and the genetic changes that underlie them ­ impact bone health. Gomez et al. aimed to address this question by focusing on KSR2, a gene involved in energy consumption and feeding behavior. Children who carry certain KSR2 mutations are prone to obesity and type 2 diabetes; mice lacking the gene also develop these conditions due to uncontrolled eating. Closely examining mutant mice in which Ksr2 had been deactivated in every cell revealed that the weight-bearing bones of these animals were also more likely to break, and the fractures then healed more slowly. This was the case even though these bones had higher mass and less marrow fat compared to healthy mice. Non-weight bearing bones (such as the spine) did not exhibit these changes. Further experiments revealed that, when expressed normally in the skeleton, Ksr2 skews the stem cell maturation process towards marrow fat cells instead of bone-creating cells. This suggests a new role for Ksr2, which therefore seems to independently regulate both feeding behavior and bone health. In addition, the work by Gomez et al. demonstrate that Ksr2 mutant mice could be a useful model to better understand how obesity and diabetes affect human bones, and to potentially develop new therapies.

Targeted Deletion of the Claudin12 Gene in Mice Increases Articular Cartilage and Inhibits Chondrocyte Differentiation.

To study the role of Claudin (CLDN)12 in bone, we developed mice with a targeted deletion of exon2 in the Cldn12 gene for skeletal phenotype analysis. Micro-CT analysis of the secondary spongiosa of distal femurs of mice with targeted disruption of the Cldn12 gene and control littermates showed no significant genotype-specific differences in either cortical or trabecular bone parameters for either gender in 13-week-old mice. Immunohistochemistry revealed that while CLDN12 was expressed in both differentiating chondrocytes and osteoblasts of the secondary spongiosa of 3-week-old wild-type mice, its expression was restricted to differentiating chondrocytes in the articular cartilage and growth plate in adult mice. Articular cartilage area at the knee were increased by 47% in Cldn12 knockout (KO) mice compared to control littermates. Micro-CT analyses found that while the trabecular number was increased by 9% and the trabecular spacing was reduced by 9% in the femoral epiphysis of Cldn12 KO mice, neither bone volume nor bone volume adjusted for tissue volume was different between the two genotypes. The expression levels of Clusterin, Lubricin and Mmp13 were increased by 56%, 46%, and 129%, respectively, in primary articular chondrocytes derived from KO compared to control mice. Our data indicate that targeted deletion of the Cldn12 gene in mice increases articular cartilage, in part, by promoting articular chondrocyte phenotype.

Monocyte Chemotactic Proteins Mediate the Effects of Hyperglycemia in Chondrocytes: In Vitro Studies.

Chemokines are secreted by a large variety of cells. They are involved in controlling cell trafficking, maturation, and differentiation. However, the specific responses and effects of chemokines on specific skeletal cell types under high glucose conditions have not been investigated. Chondrocytes play an important role in osteoarthritis and fracture healing. Delayed fracture healing is one of the major health complications caused by diabetes, so the goal of this study was to evaluate the response of several chemokines to high glucose conditions in chondrocyte cells and analyze their role in the catabolic effect of hyperglycemia. ATDC5 chondrocytes were cultured in normal and high glucose media, and mRNA expression levels of several chemokines and chondrocyte differentiation markers were quantified. Bindarit, a specific inhibitor of monocyte chemotactic proteins (MCPs), was used to determine the role of MCPs in mediating the effects of high glucose conditions in chondrocyte cells. High glucose treatment upregulated the expression of three Mcps, as well as the expression of matrix metalloproteinase 13 (Mmp13) and Osteocalcin (Oc). Furthermore, bindarit treatment downregulated Mmp13 and Oc but upregulated Collagen 2 (Col2) mRNA levels in chondrocytes treated with high glucose. Moreover, treatment of chondrocytes with ascorbic acid reduced the effect of high glucose conditions on the expression of chemokines and Mmps. These data together suggest that MCPs mediate the catabolic effect of high glucose in chondrocytes.

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton.

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (Tfr1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin, and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of Tfr1 and Tfr1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of Tfr1 in osteoclast lineage cells in vivo and in vitro, we crossed Tfrc (encoding Tfr1)-floxed mice with Lyz2 (LysM)-Cre and Cathepsin K (Ctsk)-Cre mice to generate Tfrc conditional knockout mice in myeloid osteoclast precursors (Tfr1ΔLysM) or differentiated osteoclasts (Tfr1ΔCtsk), respectively. Skeletal phenotyping by µCT and histology unveiled a significant increase in trabecular bone mass with normal osteoclast number in long bones of 10-week-old young and 6-month-old adult female but not male Tfr1ΔLysM mice. Although high trabecular bone volume in long bones was observed in both male and female Tfr1ΔCtsk mice, this phenotype was more pronounced in female knockout mice. Consistent with this gender-dependent phenomena, estrogen deficiency induced by ovariectomy decreased trabecular bone mass in Tfr1ΔLysM mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts in vitro by attenuating mitochondrial respiration and activation of the Src-Rac1-WAVE regulatory complex axis, respectively, leading to decreased bone resorption with little impact on osteoclast differentiation. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling in a gender-dependent manner.

T-cell factor 7L2 is a novel regulator of osteoblast functions that acts in part by modulation of hypoxia signaling.

T-cell-like factor (TCF)7l2, a key effector of canonical Wnt signaling, is highly expressed in bone but nothing is known about its role in regulating osteoblast function. To test this, we generated mice with conditional disruption of Tcf7l2 gene in osteoblast lineages using Tcf7l2 floxed and Col1α2-Cre mice. Skeletal parameters were evaluated using heterozygous conditional knockdown (HCKD) mice since homozygous conditional knockout died during pregnancy or immediately after birth. At 5 wk of age, trabecular bone mass of long bones was reduced by 35% as measured by microcomputed tomography (µCT). Histology data showed a 42% reduction in femur trabecular bone mass caused by reduced bone formation. Knockdown of Tcf7l2 expression in osteoblasts decreased proliferation and differentiation by 20%-40%. Expression levels of genes (Hif1α, Vegf, and ß-catenin) targeted by TCF7L2 were decreased by 50% in Tcf7l2-deficient osteoblasts and bones of HCKD mice. We found that the Hif1α gene promoter contained multiple putative TCF7L2 motifs and stabilization of HIF1α protein levels rescued expression of TCF7L2 target genes and alkaline phosphatase (ALP) activity in Tcf7l2-deficient osteoblasts. Furthermore, Tcf7l2 overexpression increased proliferation in the presence of canonical Wnt3a that was not affected by ß-catenin inhibitor providing evidence for a noncanonical signaling in mediating TCF7L2 effects. Tcf7l2 expression was increased in response to mechanical strain (MS) in vitro and in vivo, and disruption of Tcf7l2 expression in osteoblasts reduced MS-induced ALP activity by 35%. We conclude that Tcf7l2, a mechanoresponsive gene, is an important regulator of osteoblast function acting, in part, via hypoxia signaling.NEW & NOTEWORTHY TCF7L2 is expressed by bone but it was not known whether TCF7L2 expression influenced bone development. By using a mouse model with conditional disruption of Tcf7l2 in osteoblast lineage cells, we have demonstrated for the first time, that TCF7L2 plays an important role in regulating osteoblasts via a noncanonical pathway.

Role of prolyl hydroxylase domain proteins in bone metabolism.

Cellular metabolism requires dissolved oxygen gas. Because evolutionary refinements have constrained mammalian dissolved oxygen levels, intracellular oxygen sensors are vital for optimizing the bioenergetic and biosynthetic use of dissolved oxygen. Prolyl hydroxylase domain (PHD) homologs 1-3 (PHD1/2/3) are molecular oxygen dependent non-heme dioxygenases whose enzymatic activity is regulated by the concentration of dissolved oxygen. PHD oxygen dependency has evolved into an important intracellular oxygen sensor. The most well studied mechanism of PHD oxygen-sensing is its regulation of the hypoxia-inducible factor (HIF) hypoxia signaling pathway. Heterodimeric HIF transcription factor activity is regulated post-translationally by selective PHD proline hydroxylation of its HIF1α subunit, accelerating HIF1α ubiquitination and proteasomal degradation, preventing HIF heterodimer assembly, nuclear accumulation, and activation of its target oxygen homeostasis genes. Phd2 has been shown to be the key isoform responsible for HIF1α subunit regulation in many cell types and accordingly disruption of the Phd2 gene results in embryonic lethality. In bone cells Phd2 is expressed in high abundance and tightly regulated. Conditional disruption of the Phd1, Phd2 and/or Phd3 gene in various bone cell types using different Cre drivers reveals a major role for PHD2 in skeletal growth and development. In this review, we will summarize the state of current knowledge on the role and mechanism of action of PHD2 as oxygen sensor in regulating bone metabolism.

Mice with Targeted Knockout of Tetraspanin 3 Exhibit Reduced Trabecular Bone Mass Caused by Decreased Osteoblast Functions.

Tetraspanin3 (TSPAN3) was identified as a binding partner of claudin11 (CLDN11) in osteoblasts and other cell types. Mice with targeted disruption of Cldn11 exhibited trabecular bone mass deficit caused by reduced bone formation and osteoblast function. To determine if the disruption of CLDN11 interacting protein gene Tspan3 results in a similar skeletal phenotype as that of Cldn11 knockout (KO) mice, we generated homozygous Tspan3 KO and heterozygous control mice and characterized their skeletal phenotypes at 13 weeks of age. Micro-CT measurements of the secondary spongiosa of the distal femur revealed 17% and 29% reduction in trabecular bone volume adjusted for tissue volume (BV/TV) in the male and female mice, respectively. Similarly, trabecular BV/TV of the proximal tibia was reduced by 19% and 20% in the male and female mice, respectively. The reduced trabecular bone mass was caused primarily by reduced trabecular thickness and number, and increased trabecular spacing. Consistent with the reduced bone formation as confirmed by histomorphometry analyses, serum alkaline phosphatase was reduced by 11% in the KO mice as compared with controls. Our findings indicate that TSPAN3 is an important positive regulator of osteoblast function and trabecular bone mass, and the interaction of TSPAN3 with CLDN11 could contribute in part to the bone forming effects of Cldn11 in mice.

Differences in pathways contributing to thyroid hormone effects on postnatal cartilage calcification versus secondary ossification center development.

The proximal and distal femur epiphyses of mice are both weight-bearing structures derived from chondrocytes but differ in development. Mineralization at the distal epiphysis occurs in an osteoblast-rich secondary ossification center (SOC), while the chondrocytes of the proximal femur head (FH), in particular, are directly mineralized. Thyroid hormone (TH) plays important roles in distal knee SOC formation, but whether TH also affects proximal FH development remains unexplored. Here, we found that TH controls chondrocyte maturation and mineralization at the FH in vivo through studies in thyroid stimulating hormone receptor (Tshr-/-) hypothyroid mice by X-ray, histology, transcriptional profiling, and immunofluorescence staining. Both in vivo and in vitro studies conducted in ATDC5 chondrocyte progenitors concur that TH regulates expression of genes that modulate mineralization (Ibsp, Bglap2, Dmp1, Spp1, and Alpl). Our work also delineates differences in prominent transcription factor regulation of genes involved in the different mechanisms leading to proximal FH cartilage calcification and endochondral ossification at the distal femur. The information on the molecular pathways contributing to postnatal cartilage calcification can provide insights on therapeutic strategies to treat pathological calcification that occurs in soft tissues such as aorta, kidney, and articular cartilage.

Repeated mild traumatic brain injury impairs fracture healing in male mice.

OBJECTIVES: The goal of this study was to evaluate the long-term impact of repeated (r) mild traumatic brain injury (mTBI) on the healing of fractures in a mouse model. Ten week-old male mice were subjected to r-mTBI once per day for 4 days followed by closed femoral fracture using a three-point bending technique, 1 week post impact and fracture healing phenotype evaluated at 20 weeks of age. RESULTS: Micro-CT analysis of the fracture callus region at nine weeks post fracture revealed reduced bone volume (30%, p < 0.05) in the r-mTBI fracture group compared to the control-fracture group. The connectivity density of the fracture callus bone was reduced by 40% (p < 0.01) in the r-mTBI fracture group. Finite element analysis of the fracture callus region showed reduced failure load (p = 0.08) in the r-mTBI group compared to control group. There was no residual cartilage in the fracture callus region of either the r-mTBI or control fracture group. The reduced fracture callus bone volume and mechanical strength of fracture callus in r-mTBI mice 9 weeks post fracture are consistent with negative effects of r-mTBI on fracture healing over a long-term resulting in decreased mechanical strength of the fracture callus.

Lack of Skeletal Effects in Mice with Targeted Disruptionof Prolyl Hydroxylase Domain 1 (Phd1) Gene Expressed in Chondrocytes.

The critical importance of hypoxia-inducible factor (HIF)s in the regulation of endochondral bone formation is now well established. HIF protein levels are closely regulated by the prolyl hydroxylase domain-containing protein (PHD) mediated ubiquitin-proteasomal degradation pathway. Of the three PHD family members expressed in bone, we previously showed that mice with conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in the trabecular bone mass of the long bones. By contrast, loss of Phd3 expression in chondrocytes had no skeletal effects. To investigate the role of Phd1 expressed in chondrocytes on skeletal development, we conditionally disrupted the Phd1 gene in chondrocytes by crossing Phd1 floxed mice with Collagen 2α1-Cre mice for evaluation of a skeletal phenotype. At 12 weeks of age, neither body weight nor body length was significantly different in the Cre+; Phd1flox/flox conditional knockout (cKO) mice compared to Cre−; Phd1flox/flox wild-type (WT) control mice. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype differences were found for any of the trabecular bone measurements of either femur or tibia. Similarly, cortical bone parameters were not affected in the Phd1 cKO mice compared to control mice. Histomorphometric analyses revealed no significant differences in bone area, bone formation rate or mineral apposition rate in the secondary spongiosa of femurs between cKO and WT control mice. Loss of Phd1 expression in chondrocytes did not affect the expression of markers of chondrocytes (collage 2, collagen 10) or osteoblasts (alkaline phosphatase, bone sialoprotein) in the bones of cKO mice. Based on these and our published data, we conclude that of the three PHD family members, only Phd2 expressed in chondrocytes regulates endochondral bone formation and development of peak bone mass in mice.

Correction: Evaluation of CCL21 role in post-knee injury inflammation and early cartilage degeneration.

[This corrects the article DOI: 10.1371/journal.pone.0247913.].

Prolyl Hydroxylase Domain-Containing Protein 3 Gene Expression in Chondrocytes Is Not Essential for Bone Development in Mice.

We previously showed that conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in long bone trabecular bone mass. Loss of Phd2 gene expression or inhibition of PHD2 activity by a specific inhibitor resulted in a several-fold compensatory increase in Phd3 expression in chondrocytes. To determine if expression of PHD3 plays a role in endochondral bone formation, we conditionally disrupted the Phd3 gene in chondrocytes by crossing Phd3 floxed (Phd3flox/flox) mice with Col2α1-Cre mice. Loss of Phd3 expression in the chondrocytes of Cre+; Phd3flox/flox conditional knockout (cKO) mice was confirmed by real time PCR. At 16 weeks of age, neither body weight nor body length was significantly different in the Phd3 cKO mice compared to Cre-; Phd3flox/flox wild-type (WT) mice. Areal BMD measurements of total body as well as femur, tibia, and lumbar skeletal sites were not significantly different between the cKO and WT mice at 16 weeks of age. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype difference was found for any of the trabecular bone measurements of either the femur or the tibia. Trabecular bone volume of distal femur epiphysis was not different between cKO and WT mice. Histology analyses revealed Phd3 cKO mice exhibited a comparable chondrocyte differentiation and proliferation, as evidenced by no changes in cartilage thickness and area in the cKO mice as compared to WT littermates. Consistent with the in vivo data, lentiviral shRNA-mediated knockdown of Phd3 expression in chondrocytes did not affect the expression of markers of chondrocyte differentiation (Col2, Col10, Acan, Sox9). Our study found that Phd2 but not Phd3 expressed in chondrocytes regulates endochondral bone formation, and the compensatory increase in Phd3 expression in the chondrocytes of Phd2 cKO mice is not the cause for increased trabecular bone mass in Phd2 cKO mice.

Computational prediction of small molecules with predicted binding to FGFR3 and testing biological effects in bone cells.

Activating anabolic receptor-mediated signaling is essential for stimulating new bone formation and for promoting bone healing in humans. Fibroblast growth factor receptor (FGFR) 3 is reported to be an important positive regulator of osteogenesis. Presently, recombinant proteins are used to stimulate FGFR3 function but have limitations for therapy due to expense and stability. Therefore, there is a need for identification of novel small molecules binding to FGFR3 that promote biological function. In silico molecular docking and high-throughput virtual screening on zinc database identified seven compounds predicted to bind to an active site within the ßC'-ßE loop, specific to FGFR3. All seven compounds fall within an acceptable range of ADME/T properties. Four compounds showed a 30-65% oral absorption rate. Density functional theory analysis revealed a high HOMO-LUMO gap, reflecting high molecular stability for compounds 14977614 and 13509082. Five compounds exhibited mutagenicity, while the other three compounds presented irritability. Computational mutagenesis predicted that mutating G322 affected compound binding to FGFR3. Molecular dynamics simulation revealed compound 14977614 is stable in binding to FGFR3. Furthermore, compound 14977614, with an oral absorption rate of 60% and high molecular stability, produced significant increases in both proliferation and differentiation of bone marrow stromal cells in vitro. Anti-FGFR3 treatment completely blocked the stimulatory effect of 14977614 on BMSC proliferation. Ex vivo treatment of mouse calvaria in organ culture for seven days with 14977614 increased mineralization and expression levels of bone formation markers. In conclusion, computational analyses identified seven compounds that bind to the FGFR3, and in vitro studies showed that compound 14977614 exerts significant biological effects on osteogenic cells.