Sarah L. Dallas

Von Kossa Staining Alone Is Not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation

Numerous techniques are currently used to characterize biological mineralization in intact tissues and cell cultures; the von Kossa staining method, electron microscopic analysis (EM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR) are among the most common. In this study, we utilized three of these methods to compare the mineralization of cultured fetal rat calvarial cells (FRC) and the osteoblast cell lines 2T3 and MC3T3-E1 with the in vivo mineral of rat calvarial bone. The cells were cultured with or without ascorbic acid (100 µg/ml) and β-glycerophosphate (2.5, 5, or 10 mM βGP), and harvested between 16 and 21 days (FRC cells and 2T3 cells) or at 30 days of culture (MC3T3-E1 cells). In the FRC cultures, maximal von Kossa staining was observed with 2.5 and 5 mM βGP in the presence of 100 µg/ml ascorbate. FRC cells also showed some von Kossa staining when cultured with βGP alone. In contrast, maximal von Kossa staining for MC3T3-E1 cells was observed with 10 mM βGP. Only the cultures of MC3T3-E1 cells that received both ascorbate and βGP produced von Kossa positive structures. The 2T3 cultures produced von Kossa positive staining only upon treatment with ascorbic acid and βGP, which was greatly accelerated by bone morphogenic protein-2 (BMP-2). FTIR was performed on the mineral and matrix generated in FRC, MC3T3, and 2T3 cultures, and the results were compared with spectra derived from 16-day-old rat calvaria. The mineral-to-matrix ratios calculated from FTIR spectra for rat calvaria ranged from 2.97 to 7.44. FRC cells made a bonelike, poorly crystalline apatite, and, with increasing βGP, there was a statistically significant (P ≤ 0.02) dose-dependent increase in the mineral-to-matrix ratio (0.56 ± 0.16, 1.00 ± 0.32, and 2.46 ± 0.76, for 2.5, 5, and 10 mM βGP, respectively). The mean carbonate-to-phosphate ratios of the FRC cultures were 0.015, 0.012, and 0.008, in order of increasing βGP concentration, compared with rat calvaria values of 0.009–0.017. The 2T3 cells treated with BMP-2 also made bonelike crystals, similar to those observed in FRC cultures. In contrast, the cultures of von Kossa positive MC3T3-E1 cells did not display a significant amount of mineral (maximum mineral-to-matrix ratio was 0.4). Thus, although the von Kossa stainings of FRC, 2T3, and MC3T3-E1 were very similar, FTIR analysis indicated that calcium phosphate mineral was not present in the MC3T3 cultures. By EM, the mineral in FRC cell cultures and 2T3 cultures was generally associated with collagen, whereas rare or sparse dystrophic mineralization of unknown chemical origin was evident in the MC3T3-E1 cultures. These studies demonstrate that von Kossa staining alone is not appropriate for the identification and quantitation of bonelike mineral and, hence, other techniques such as X-ray diffraction, EM, or FTIR should be utilized to verify the presence and quality of calcium phosphate phases.

BONE MORPHOGENETIC PROTEIN 2 (BMP-2) ENHANCES BMP-3, BMP-4, AND BONE CELL DIFFERENTIATION MARKER GENE EXPRESSION DURING THE INDUCTION OF MINERALIZED BONE MATRIX FORMATION IN CULTURES OF FETAL RAT CALVARIAL OSTEOBLASTS

Normal bone formation is a prolonged process that is carefully regulated and involves sequential expression of growth regulatory factors by osteoblasts as they proliferate and ultimately differentiate. Since this orderly sequence of gene expression by osteoblasts suggests a cascade effect, and BMP-2 is capable of initiating and maintaining this effect, we examined the effects of BMP-2 on expression of other BMPs and compared these effects with the expression pattern of bone cell differentiation marker genes in primary cultures of fetal rat calvarial (FRC) osteoblasts. To examine the gene expression profile during bone cell differentiation and bone formation, we also examined the effects of rBMP-2 on bone formation in vivo and in vitro. rBMP-2 stimulated bone formation on the periosteal surface of mice when 500 ng/day rBMP-2 was injected subcutaneously. When rBMP-2 was added to primary cultures of FRC osteoblasts, it accelerated mineralized nodule formation in a time and concentration-dependent manner (10–40 ng/ml). rBMP-2 (40 ng/ml) enhanced BMP-3 and -4 mRNA expression during the mineralization phase of primary cultures of FRC osteoblasts. Enhancement of BMP-3 and -4 mRNA expression by rBMP-2 was associated with increased expression of bone cell differentiation marker genes, alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), osteopontin (OP), and bone sialoprotein (BSP). These results suggest that BMP-2 enhances expression of other BMP genes during bone cell differentiation. BMP-2 may act in a paracrine fashion in concert with other BMPs it induces to stimulate bone cell differentiation and bone formation during remodeling.

Fibrillin-1 regulates the bioavailability of TGFβ1

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) β1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFβ signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44–49 releases endogenous TGFβ1, thereby stimulating TGFβ receptor–mediated Smad2 signaling. This altered TGFβ1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFβ1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFβ1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFβ-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44–49 can contribute to MFS and related diseases.

The effects of cytokines and growth factors on osteoblastic cells

In this short review, some regulatory mechanisms that are involved in the control of normal bone formation are proposed, based on several in vivo and in vitro models our group has utilized recently to study osteoblast differentiation and mineralized bone matrix formation. Of course, these proposals must be assessed in the light of the limitations of the models, which probably represent a simplification of the complex and different ways in which normal mammalian bone is formed at different sites. Nevertheless, it is likely that the same general types of control mechanisms are active in each of the different types of bone formation. In adult humans, bone formation predominantly occurs by remodeling, the process by which bone which has recently been resorbed by osteoclasts is replaced by teams of osteoblasts. Other types of bone formation such as endochondral bone formation and appositional bone formation are also important, particularly during growth and adolescence. The end results of each of these processes are the same, namely a complex mineralized proteinaceous bone matrix. These processes are modulated by systemic hormonal influences, which are particularly important with respect to pituitary hormones and sex steroids during growth and adolescence, and by local cellular microenvironmental differences. The former will not be discussed here. Rather, we will concentrate on the local events and factors which are likely involved in the bone formation process occurring during normal bone remodeling.

A murine model of human myeloma bone disease

Myeloma causes a devastating and unique form of osteolytic bone disease. Although osteoclast activation is responsible for bone destruction, the precise mechanisms by which myeloma cells increase osteoclast activity have not been defined. An animal model of human myeloma bone disease would help in clarification of these mechanisms. Multiple myeloma occurs spontaneously in aging C57 BL/KaLwRij mice and has all of the features of the disease in humans, including the characteristic bone lesions. The disease can be induced in normal C57 BL/KaLwRij mice by inoculation of fresh marrow-derived cells from mice with myeloma, but this model is difficult to study because of variability in the number of myeloma cells in marrow-derived preparations. To develop a better animal model of human myeloma bone disease, we have established and subcloned a cell line from this murine myeloma and found that it causes osteolytic bone lesions in mice characteristic of human myeloma bone disease. The cell line produces interleukin-6, but grows independent of exogenous interleukin-6. Mice inoculated intravenously with the cultured cells predictably develop an identical disease to the mice injected intravenously with fresh bone-marrow-derived myeloma cells, including monoclonal gammopathy and radiologic bone lesions. We found that some of the mice became hypercalcemic, and the bone lesions are characterized by increased osteoclast activity. We found identical results when we inoculated Nu/Bg/XID mice with cultured murine myeloma cells. Because we can inoculate mice with precise numbers of cells and predict accurately when the mice will develop bone lesions, become hypercalcemic, and die, this should be a convenient model for determining the mechanisms by which the myeloma cells cause osteoclast activation in this model of human myeloma bone disease.

Potential Role for Heparan Sulfate Proteoglycans in Regulation of Transforming Growth Factor-β (TGF-β) by Modulating Assembly of Latent TGF-β-binding Protein-1

Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-β in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-β 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-β into the ECM, with increased amounts of soluble latent TGF-β. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by β-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-β availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin.

HtrA1 inhibits mineral deposition by osteoblasts - Requirement for the protease and PDZ domains

HtrA1 is a secreted multidomain protein with serine protease activity. In light of increasing evidence implicating this protein in the regulation of skeletal development and pathology, we investigated the role of HtrA1 in osteoblast mineralization and identified domains essential for this activity. We demonstrate increased HtrA1 expression in differentiating 2T3 osteoblasts prior to the appearance of mineralization. HtrA1 is subsequently down-regulated in fully mineralized cultures. The functional role of HtrA1 in matrix calcification was investigated using three complementary approaches. First, we transfected a full-length HtrA1 expression plasmid into 2T3 cells and showed that overexpression of HtrA1 delayed mineralization, reduced expression of Cbfa1 and collagen type I mRNA, and prevented BMP-2-induced mineralization. Second, knocking down HtrA1 expression using short interfering RNA induced mineral deposition by 2T3 cells. Third, by expressing a series of recombinant HtrA1 proteins, we demonstrated that the protease domain and the PDZ domain are essential for the inhibitory effect of HtrA1 on osteoblast mineralization. Finally, we tested whether HtrA1 cleaves specific matrix proteins that are known to regulate osteoblast differentiation, mineralization, and/or BMP-2 activity. Full-length recombinant HtrA1 cleaved recombinant decorin, fibronectin, and matrix Gla protein. Both the protease domain and the PDZ domain were necessary for the cleavage of matrix Gla protein, whereas the PDZ domain was not required for the cleavage of decorin or fibronectin. Type I collagen was not cleaved by recombinant HtrA1. These results suggest that HtrA1 may regulate matrix calcification via the inhibition of BMP-2 signaling, modulating osteoblast gene expression, and/or via the degradation of specific matrix proteins.

Growth Regulatory Factors and Bone

All of the diseases of bone occur because of disruption of the normal process by which bone is remodeled. The normal bone remodeling process in adults occurs in discrete and localized packets throughout the skeleton, and is comprised of the coupled processes of bone resorption and bone formation. The sequence is always the same, whether it occurs in the Haversian systems of cortical bone or on the trabecular surfaces of compact bone, and begins with a short phase of osteoclastic bone resorption (7±10 days), followed by a prolonged phase of bone formation (2±3 months). Since the remodeling sequence is dependent on the functions of distinct and separate populations of cells (osteoclasts and osteoblasts), it is apparent that for balance and coordination between the activities of these cell populations, there must be a complex but highly integrated system of extracellular signals which are responsible. Since the bone formation phase of the remodeling sequence is so prolonged, and results in a team of osteoblasts completely ®lling in the resorption bays formed during the phase of osteoclastic resorption, it is clear that the process must be highly coordinated with bone resorption and tightly regulated by local signals or factors generated as a consequence of the resorption process. The phase of bone formation is comprised of a discrete series of cellular events, which include chemotaxis of osteoblast precursors to the sites of resorption defects, proliferation of these precursors to form a team of osteoblasts capable of ®lling in the resorption defect, and then differentiation of the preosteoblasts to form mature cells which are responsible for expressing the structural proteins of bone such as type-1 collagen and other functional proteins such as alkaline phosphatase and osteocalcin. We are now gaining considerable insights into how these cellular events are coordinated by the growth regulatory factors which are produced in the microenvironment of the remodeling site. Osteoblasts are cells derived from pluripotent mesenchymal precursors. As they proliferate and differentiate, these cells express growth regulatory factors which are responsible for controlling the whole process. Some such as the ®broblast growth factors (FGFs), transforming growth factor b (TGFb) and insulin-like growth factors (IGFs) are primarily responsible for the initial events involved in the remodeling process, and predominantly for enhancing proliferation of osteoblast precursors. Others, namely the bone morphogenetic protein (BMP) family, are responsible for osteoblast differentiation and matrix formation. These growth regulatory factors are expressed by osteoblasts, but some at least also by osteoclasts [1]. These growth factors are also present in the bone matrix itself and released in active form during the process of bone resorption [2,3]. This has been clearly demonstrated for TGFb [3], but is also likely true for the other growth factors as well. These growth regulatory factors, present in abundant supply locally, are then presumably available to regulate all of the cellular events involved in the remodeling process. The availability of these growth regulatory factors at the resorption site has important implications not only for bone remodeling in the formation phase of the normal remodeling process, but also possibly for the repair of bone following fracture or injury. It is also likely that these growth factors may under some circumstances play a harmful role. For example, they may, when present locally in the bone microenvironment, affect the behavior of metastatic cancer cells such as breast cancers. Yin et al. [4] have shown that breast cancer

Crosstalk Between MLO-Y4 Osteocytes and C2C12 Muscle Cells Is Mediated by the Wnt/β-Catenin Pathway: WNT/β-CATENIN PATHWAY MEDIATES OSTEOCYTES AND MUSCLE CELLS CROSSTALK

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO‐Y4 osteocyte‐like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ∼25%. MLO‐Y4 and primary osteocyte CM (1% to 10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Because WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO‐Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5 ng/mL mirrored the effects of both primary osteocyte and MLO‐Y4 CM by inducing nuclear translocation of β‐catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO‐Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100 ng/mL) inhibited both the effects of MLO‐Y4 CM and WNT3a on C2C12 cell differentiation. RT‐PCR array results supported the activation of the Wnt/β‐catenin pathway by MLO‐Y4 CM and WNT3a. These results were confirmed by qPCR, showing upregulation of myogenic markers and two Wnt/β‐catenin downstream genes, Numb and Flh1. We postulated that MLO‐Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO‐Y4 CM and WNT3a increased caffeine‐induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt/ β‐Catenin pathway.

Absence of herpesvirus DNA sequences in the 5T murine model of human multiple myeloma

Kaposis sarcoma‐associated herpesvirus (KSHV, also known as HHV‐8) has been found in patients with multiple myeloma (MM) and postulated to be aetiologically associated with the development of this common plasma cell malignancy. A murine model of MM was previously established in which intravenous transfer of 5T myeloma cells into C57BL/KaLwRij mice resulted in characteristic features of human MM. In the present study, we sought to identify herpesvirus DNA sequences in this murine model of MM through polymerase chain reaction (PCR) analysis using primers specific for KSHV, murine herpesvirus 68 (MHV68) and murine cytomegalovirus (MCMV) as well as consensus primers designed from the highly conserved DNA polymerase genes of the Herpesviridae family. None of the DNA samples from whole bone marrow (n = 6) or dendritic cells enriched by long‐term culture (n = 8) of 5T myeloma‐bearing mice as well as the 5T myeloma cell lines (n = 3) maintained in long‐term culture yielded specific amplification products in any of the PCR assays. Two KSHV‐specific serological assays measuring antibodies to KSHV latent and lytic antigens also failed to detect the presence of anti‐KSHV antibodies in mice that developed MM. These results suggest that the development of 5T murine MM is unlikely to be involved with KSHV or a KSHV‐like murine herpesvirus.