Steven N. Popoff

Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development

Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf-/- mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.

Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo.

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix‐associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti‐CTGF neutralizing antibody caused a dose‐dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N‐linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3‐ to 4‐fold overexpressed in op versus normal bone. RT‐PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non‐osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast‐specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function. J. Cell. Biochem. 84: 12–26, 2002.

The osteopetrotic mutation toothless (tl) is a loss-of-function frameshift mutation in the rat Csf1 gene: Evidence of a crucial role for CSF-1 in osteoclastogenesis and endochondral ossification

The toothless (tl) mutation in the rat is a naturally occurring, autosomal recessive mutation resulting in a profound deficiency of bone-resorbing osteoclasts and peritoneal macrophages. The failure to resorb bone produces severe, unrelenting osteopetrosis, with a highly sclerotic skeleton, lack of marrow spaces, failure of tooth eruption, and other pathologies. Injections of CSF-1 improve some, but not all, of these. In this report we have used polymorphism mapping, sequencing, and expression studies to identify the genetic lesion in the tl rat. We found a 10-base insertion near the beginning of the open reading of the Csf1 gene that yields a truncated, nonfunctional protein and an early stop codon, thus rendering the tl rat CSF-1null. All mutants were homozygous for the mutation and all carriers were heterozygous. No CSF-1 transcripts were identified in rat mRNA that would avoid the mutation via alternative splicing. The biology and actions of CSF-1 have been elucidated by many studies that use another naturally occurring mutation, the op mouse, in which a single base insertion also disrupts the reading frame. The op mouse has milder osteoclastopenia and osteopetrosis than the tl rat and recovers spontaneously over the first few months of life. Thus, the tl rat provides a second model in which the functions of CSF-1 can be studied. Understanding the similarities and differences in the phenotypes of these two models will be important to advancing our knowledge of the many actions of CSF-1.

Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function

Osteoactivin (OA) is a novel glycoprotein that is highly expressed during osteoblast differentiation. Using Western blot analysis, our data show that OA protein has two isoforms, one is transmembranous and the other is secreted into the conditioned medium of primary osteoblasts cultures. Fractionation of osteoblast cell compartments showed that the mature, glycosylated OA isoform of 115 kDa is found in the membranous fraction. Both OA isoforms (secreted and transmembrane) are found in the cytoplasmic fraction of osteoblasts. Overexpression of EGFP-tagged OA in osteoblasts showed that OA protein accumulates into vesicles for transportation to the cell membrane. We examined OA protein production in primary osteoblast cultures and found that OA is maximally expressed during the third week of culture (last stage of osteoblast differentiation). Glycosylation studies showed that OA isoform of 115 kDa is highly glycosylated. We also showed that retinoic acid (RA) stimulates the mannosylation of OA protein. In contrast, tunicamycin (TM) strongly inhibited N-glycans incorporation into OA protein. The functional role of the secreted OA isoform was revealed when cultures treated with anti-OA antibody, showed decreased osteoblast differentiation compared to untreated control cultures. Gain-of-function in osteoblasts using the pBABE viral system showed that OA overexpression in osteoblast stimulated their differentiation and function. The availability of a naturally occurring mutant mouse with a truncated OA protein provided further evidence that OA is an important factor for terminal osteoblast differentiation and mineralization. Using bone marrow mesenchymal cells derived from OA mutant and wild-type mice and testing their ability to differentiate into osteoblasts showed that differentiation of OA mutant osteoblasts was significantly reduced compared to wild-type osteoblasts. Collectively, our data suggest that OA acts as a positive regulator of osteoblastogenesis.

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-β1 to induce mesenchymal cell condensation

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor β1 (TGF‐β1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF‐β1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF‐β1 stimulation concomitant with dramatic up‐regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF‐β1‐induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up‐regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF‐β1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF‐β1 and FN were co‐expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF‐β1‐induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up‐regulating FN and suppressing Sox9 expression during TGF‐β1 induced MC condensation. J. Cell. Physiol. 210: 398–410, 2007.

Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function

Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast‐related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP‐2 and the role OA plays as a downstream mediator of BMP‐2 effects in osteoblast function. Using primary osteoblast cultures, we tested different doses of BMP‐2 on the regulation of OA expression during osteoblast development. To test whether Smad‐1 signaling is responsible for BMP‐2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP‐2 and analyzed by Western blot. BMP‐2 treatment increased OA mRNA and protein expression in a dose‐dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. We next examined whether the role of OA as a downstream mediator of BMP‐2 effects on osteoblast differentiation and matrix mineralization. Osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP‐2. Cultures transfected with OA antisense oligonucleotides and treated with BMP‐2 showed a reduction of OA expression associated with a significant reduction in early and late differentiation markers induced by BMP‐2. Therefore, OA acts, at least in part, as a downstream mediator of BMP‐2 effects on osteoblast differentiation and matrix mineralization. Our findings suggest that BMP‐2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP‐2 effects on osteoblast differentiation and function. J. Cell. Physiol. 210: 26–37, 2007.

Serum and Tissue Cytokines and Chemokines Increase with Repetitive Upper Extremity Tasks

We investigated inflammation in rats performing a low repetition, negligible force (LRNF) or high repetition, negligible force (HRNF) task of reaching and retrieving food pellets at target rates of two or four reaches/min for 2 h/day, for 6–8 weeks. Serum was assayed for 11 cytokines and chemokines; forelimb tissues for four cytokines. Macrophages were counted in forelimb tissues of LRNF rats to add to results from our previous studies of HRNF rats. In HRNF rats, serum IL‐1α, IL‐1β, TNFα, MIP2, MIP3a, and RANTES were elevated in weeks 6 and 8. In contrast, only MIP2 and MIP3a increased in serum of LRNF rats. In 8 week HRNF reach limb tissues, IL‐1α, IL‐1β, TNFα, and IL‐10 increased in distal bones, IL‐1α and ‐β in muscles, and TNFα in tendons. Only IL‐10 increased in LRNF reach limb muscles in week 8. Serum IL‐1α and MIP2 correlated with macrophages in LRNF loose connective tissues, serum MIP3a and MIP2 correlated negatively with grip strength, while serum TNFα, MIP3a, and MIP2 correlated positively with total number of reaches. Thus, several tissue and circulating cytokines/chemokines increase in an exposure dependent manner following short‐term performance of repetitive reaching tasks and correlate with macrophage infiltration and decreasing grip strength.

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine‐rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor‐β1 (TGF‐β1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF‐β1‐induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF‐β1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF‐β1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF‐specific siRNA was used to prevent TGF‐β1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up‐regulation. To examine the effects of TGF‐β1 on osteoblast cell growth, cultures were treated with TGF‐β1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF‐β1‐induced cell‐cycle arrest. Cultures transfected with CTGF siRNA prior to TGF‐β1 treatment showed an even greater reduction in cell number, suggesting that TGF‐β1‐induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF‐β1‐induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation. J. Cell. Physiol. 210: 843–852, 2007.

Experimental studies of osteopetrosis in laboratory animals

Osteopetrosis is a metabolic bone disease characterized by a systemic increase in skeletal mass. It results from a defect in the production or function of osteoclasts and is inherited in nine genetically distinct osteopetrotic animal mutations and man. Studies of these mutations have revealed that osteopetrosis is a complex, heterogeneous disorder in its expression, etiology, and response to treatment by bone marrow transplantation or by hormone/growth factor therapy. These animal mutations have been valuable tools for probing the pathogenesis and treatment of osteopetrosis, and information obtained from these studies has been used clinically for the treatment of humans with osteopetrosis. In addition, studies of these mutations have contributed significantly to understanding normal bone cell biology, including the origin of the osteoclast and the significance of colony-stimulating factor-1 in osteoclast development. The resistance of some of these mutations to cure by stem cell transplantation and hormone therapy, coupled with similar observations and experiences in the human condition, indicates that these animal mutations will continue to serve important roles in the development of alternative therapies to treat resistant forms of the disease. These studies are bound to improve the understanding of normal bone biology by providing additional insights into the regulation of osteoclasts by osteoblasts and their products or by other elements of the skeletal microenvironment.