P2Y(2) receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes.

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX-2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y(2) mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y(2) and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction.

Soluble perlecan domain I enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells.

BACKGROUND: Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro. RESULTS: In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2. CONCLUSIONS: Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.

Short-term intermittent PTH 1-34 administration enhances bone formation in SCID/Beige mice.

The anabolic effect of intermittent PTH on bone is variable depending on the species studied, duration/mode of administration, and location of skeletal response investigated. We tested the hypothesis low dose, short term, intermittent PTH 1-34 administration is sufficient to enhance bone formation without altering bone resorption. To test our hypothesis, mice were treated intermittently with one of three concentrations of PTH 1-34 (1 microg/kg; low, 10 microg/kg, or 20 microg/kg; high) for three weeks. The skeletal response was identified by quantifying: serum markers of bone turnover, cancellous bone parameters in distal femur, proximal tibia, and lumbar vertebrae by microCT, and number of osteoblasts and osteoclasts in distal femur. Mice receiving 20 microg/kg of PTH 1-34 demonstrated a 30% increase in serum osteocalcin, but no differences in serum calcium, type I collagen teleopeptides, or TRACP 5b. For all bones, microCT analysis suggested mice receiving 20 microg/kg of PTH 1-34 had increased cancellous bone mineral density, trabecular thickness and spacing, but decreased trabecular number. A 60% increase in the number of alkaline phosphatase positive osteoblasts in the distal femur was also observed in tissue sections; however, the number of TRAP positive osteoclasts was not different between test and control groups. While animals administered 10 microg/kg demonstrated similar trends for all bone turnover indices, such alterations were not observed in animals administered PTH 1-34 at 1 microg/kg per day. Thus, PTH 1-34, administered intermittently for three weeks at 20 microg/kg is sufficient to enhance bone formation without enhancing resorption.

Osteosclerotic prostate cancer metastasis to murine bone are enhanced with increased bone formation.

Spontaneous development of osteoblastic lesions of prostate cancer (PCa) in mice is modeled by orthotopic (intraprostatic) deposition of neoplastic cells followed by an extremely long latency associated with low incidence of spontaneous bone metastasis. Intracardial injection results in overt bone metastases only with osteoclastic PCa cells (i.e., PC-3). Herein, we report that androgen independent osteoblastic PCa cells readily colonize bone when in a high remodeling state. SCID/Beige mice were subjected to periods of intermittent human parathyroid hormone 1-34 (hPTH) exposure, followed by an intracardiac infusion of osteoblastic C4-2 PCa cells. At the time of PCa infusion, analysis of bone turnover markers from mice treated with hPTH revealed significant increases in osteocalcin (55.06 +/- 7.5 vs. 74.01 +/- 18.5 ng/ml) and TRAcP-5b (3.3 +/- 0.6 vs. 4.81 +/- 0.8 U/l), but no change in type I collagen C-terminal teleopeptide levels relative to control mice. Analysis of femoral cancellous bone architecture revealed significant increases in bone mineral density, trabecular thickness (0.056 +/- 0.002 vs. 0.062 +/- 0.001 mm) and porosity, but significant decreases in connectivity density and trabecular number in hPTH treated mice relative to controls. By 8 weeks post-infusion, 70% of mice pre-treated with hPTH demonstrated detectable serum prostate specific antigen (PSAs) ranging between 2 and 18.8 ng/ml. Immuno-histochemical labeling of femurs for PSA and pan-Cytokeratin revealed the presence of significant tumor cell nests in marrow and trabecular spaces. These results suggest that: (1) local bone physiology is an important factor for developing osteoblastic/sclerotic PCa bone metastases in murine hosts; (2) the establishment of osteosclerotic PCa bone metastases in mice is enhanced by alterations that drive bone formation.

Potential role for heparan sulfate proteoglycans in regulation of transforming growth factor-beta (TGF-beta) by modulating assembly of latent TGF-beta-binding protein-1.

Latent transforming growth factor-beta-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-beta in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-beta into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-beta into the ECM, with increased amounts of soluble latent TGF-beta. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by beta-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-beta availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.

Chondrogenic differentiation on perlecan domain I, collagen II, and bone morphogenetic protein-2-based matrices.

Extracellular matrix (ECM) molecules in cartilage cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). We hypothesized that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on 2-dimensional P-C fibrils or 3-dimensional P-C/BMP-2-coated (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II, and aggrecan. Immunostaining for aggrecan, perlecan, tenascin, and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We conclude that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenic differentiation.

Spatiotemporal distribution of heparan sulfate epitopes during murine cartilage growth plate development.

Heparan sulfate proteoglycans (HSPGs) are abundant in the pericellular matrix of both developing and mature cartilage. Increasing evidence suggests the action of numerous chondroregulatory molecules depends on HSPGs. In addition to specific functions attributed to their core protein, the complexity of heparan sulfate (HS) synthesis provides extraordinary structural and functional heterogeneity. Understanding the interactions of chondroregulatory molecules with HSPGs and their subsequent outcomes has been limited by the absence of a detailed analysis of HS species in cartilage. In this study, we characterize the distribution and variety of HS species in developing cartilage of normal mice. Cryo-sections of femur and tibia from normal mouse embryos were evaluated using immunostaining techniques. A panel of unique phage display antibodies specific to particular HS species were employed and visualized with secondary antibodies conjugated to Alexa-fluor dyes. Confocal microscopy demonstrates that HS species are dynamic structures within developing growth plate cartilage and the perichondrium. GlcNS6S-IdoUA2S-GlcNS6S species are down regulated and localization of GlcNS6S-IdoUA-GlcNS6S species within the hypertrophic zone of the growth plate is lost during normal development. Regional differences in HS structures are present within developing growth plates, implying that interactions with and responses to HS-binding proteins also may display regional specialization.

Ribozyme-mediated perlecan knockdown impairs chondrogenic differentiation of C3H10T1/2 fibroblasts.

Perlecan (Pln) is an abundant heparan sulfate (HS) proteoglycan in the pericellular matrix of developing cartilage, and its absence dramatically disrupts endochondral bone formation. This study examined two previously unexamined aspects of the function of Pln in mesenchymal chondrogenesis in vitro. Using the well-established high-density micromass model of chondrogenic differentiation, we first examined the requirement for endogenous Pln synthesis and secretion through the use of Pln-targeted ribozymes in murine C3H10T1/2 embryonic fibroblasts. Second, we examined the ability of the unique N-terminal, HS-bearing Pln domain I (PlnDI) to synergize with exogenous bone morphogenetic protein-2 (BMP-2) to support later stage chondrogenic maturation of cellular condensations. The results provide clear evidence that the function of Pln in late stage chondrogenesis requires Pln biosynthesis and secretion, because 60%-70% reductions in Pln greatly diminish chondrogenic marker expression in micromass culture. Additionally, these data support the idea that while early chondrocyte differentiation can be supported by exogenous HS-decorated PlnDI, efficient late stage PlnDI-supported chondrogenesis requires both BMP-2 and Pln biosynthesis.

Heparan sulfate proteoglycans: coordinators of multiple signaling pathways during chondrogenesis.

Heparan sulfate proteoglycans are abundantly expressed in the pericellular matrix of both developing and mature cartilage. Increasing evidence indicates that the action of numerous chondroregulatory molecules depends on these proteoglycans. This review summarizes the current understanding of the interactions of heparan sulfate chains of cartilage proteoglycans with both soluble and nonsoluble ligands during the process of chondrogenesis. In addition, the consequences of mutating genes encoding heparan sulfate biosynthetic enzymes or heparan sulfate proteoglycan core proteins on cartilage development are discussed.

Perlecan functions in chondrogenesis: insights from in vitro and in vivo models.

Perlecan is a large heparan sulfate proteoglycan that is typically found in basal lamina of adult and embryonic tissues. Recent studies have demonstrated that perlecan accumulates impressively during cartilage development and is maintained as the major heparan sulfate proteoglycan of adult cartilage. In vertebrates, perlecan mutations result in skeletal defects. Moreover, in vitro studies indicate that perlecan can stimulate early stages of cartilage differentiation and cooperate with chondrogenic growth factors to promote this process. This short article will summarize these results and propose a model for perlecan function that incorporates these genetic and cell biological findings.

Perlecan-stimulated nodules undergo chondrogenic maturation in response to rhBMP-2 treatment in vitro.

The heparan sulfate proteoglycan, perlecan, is localized to hypertrophic chondrocytes in the growth plates of long bones. Mice mutants for perlecan display severe cartilage and skeletal defects. Previously, we demonstrated that C3H10T1/2 fibroblasts cultured on perlecan stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stain intensely with Alcian blue, and antibodies specific for collagen type II and aggrecan; however, nodules do not express collagen type X, a marker of chondrogenic maturation. In this investigation, we tested the hypothesis that addition of rhBMP-2 to perlecan-induced nodules would promote chondrogenic maturation in vitro. C3H10T1/2 fibroblasts were seeded in Lab-Tek chambered "Permanox" slides uncoated or coated with perlecan (B&D, 5 microg/well), at a density of 2 x 10(5) cells/well. The cells were maintained in CMRL-1066 media supplemented with ascorbic acid, citrate, and pyruvate (50 ng/ml). C3H10T1/2 fibroblasts seeded on perlecan-coated wells began to condense and form cell aggregates within 15 min. On the third day postplating, the media was replaced and supplemented with or without rhBMP-2 (50 ng/ml, Genetics Institute). On day 6 of culture, microscopy revealed that rhBMP-2-treated cultures had significantly proliferated; however, untreated cultures had not. By day 12 of culture, confocal microscopy revealed that perlecan-stimulated nodules treated with rhBMP-2 express a late stage marker of chondrogenesis (collagen type X). Morphologically, cells expressing collagen type X in rhBMP-2-treated nodules appear larger in diameter, relative to cells not expressing collagen type X. Cells cultured on plastic and treated with rhBMP-2 did not form nodules, but attached and spread, yielding a high-density monolayer. In response to rhBMP-2 treatment, these cells also express collagen type X. However, the appearance of collagen type X occurs at a later time point relative to the appearance of collagen type X in perlecan-stimulated nodules. Thus, perlecan-stimulated nodules do mature at a faster rate when treated with rhBMP-2 relative to monolayer cells.

Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I.

C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pln molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln I(mut)) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pln IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 microm on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pln I-based polypeptides lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pln IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pln provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.