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7. Regulation of growth plate chondrocytes and bone cells

In addition to their direct effects on osteogenic cells that will be discussed in a later paper, systemic agents may have important interactions with local factors in bone and cartilage. Local factors are necessary for intercellular communication and include cytokines and growth factors. A cytokine can be defined as a soluble low molecular weight cell product that affects the activity of other local cells in a paracrine manner; they may act on their cell of origin by an autocrine mechanism, or via release into the circulation may affect cells at a distant site, behaving as classic endocrine agents. In hard tissues another mechanism of control exists, where locally produced growth factors, or those in the circulation, are incorporated into mineralised matrix and are released during matrix dissolution by osteoclasts or chondroclasts. The term cytokine is now generally used to include molecules that were originally defined as growth factors, e.g., the insulin-like growth factors (IGFs), the transforming growth factors (TGF alpha and TGF beta), platelet-derived growth factor (PDGF), and fibroblast growth factors (FGFs). The list of known cytokines is ever increasing and the availability of recombinant forms has allowed extensive study of their biological activities.

In recent years it has been demonstrated that a large number of growth factors and cytokines regulate the proliferation and differentiation of bone and cartilage cells in vitro and in vivo (Table 2). This subject has been extensively reviewed (Goldring & Goldring, 1990; Canalis, McCarthy & Centrella, 1988a; Price & Russell, 1992; Martin, 1989). There is also increasing evidence that abnormal production of cytokines in diseases such as rheumatoid arthritis, osteoarthritis and osteoporosis may result in inappropriate responses by bone and cartilage cells. Those cytokines and growth factors considered to be of particular importance during bone development and growth include the IGFs, TGF a and b, bone morphogenetic proteins (BMPs), FGF, PDGF and epidermal growth factor (EGF). Many of the cell types present in the microenvironment of growing bone contribute to the local synthesis of cytokines and growth factors including the resident endothelial cells, marrow stromal cells, osteoblasts, periosteal cells and chondrocytes. The haemopoetic cells present in bone marrow include circulating monocytes, macrophages and T cells; these are another potential source of cytokines. In fact, several lines of evidence point to there being a close relationship between bone cells and cells of the immune system (Skjodt & Russell, 1993).

Table 2. Local mediators in skeletal tissues

7.1. Insulin-like growth factors (IGF-I & IGF-II)

Of the growth factors, those with the most potent effects on growing skeletal tissue are the IGFs, previously known as somatomedins. IGFs are synthesised in the liver and circulate bound to carrier proteins (Froesch et al., 1985). The major factors regulating IGF concentrations in serum are growth hormone, nutritional intake and thyroid hormones, the latter being necessary for growth hormone secretion. The traditional view was that growth hormone acted indirectly on the growth plate via IGF-I, a potent mitogen for growth plate chondrocytes. However, there is increasing evidence that growth hormone has direct effects on the growth plate (Tripper et al., 1989). (This close relationship between circulating IGFs and growth hormone will be discussed in more detail in the chapter covering the hormonal regulation of growth.)

In addition to having effects on the growth plate chondrocytes, locally synthesised and circulating IGFs retained in bone matrix are important in the regulation of bone remodelling. Osteoblasts synthesise IGFs; with human bone cells producing more IGF-II relative to IGF-I, and in human bone matrix IGF-II is present in 10-15-fold greater concentrations than IGF-I (Canalis, McCarthy & Centrella, 1988b; Mohan et al., 1988). However, in the rat, IGF-I is considered to be more important than IGF-II in regulating bone cell metabolism (Mohan & Baylink, 1991). Both IGF-I and IGF-II stimulate osteoblast and chondrocyte proliferation, induce differentiation in osteoblasts and maintain the chondrocyte phenotype (McCarthy, Centrella & Canalis, 1989; Sandell et al., 1988). Some of the anabolic effects of PTH and oestrogen on bone may be effected by alterations in the local synthesis of IGFs (Canalis et al., 1988; Ernst, Heath & Rodan, 1989). Local concentrations of IGFs will also be regulated by osteoblastic synthesis of binding proteins (IGFBPs), IGFBPs synthesis itself being altered by growth hormone and oestradiol (Schmid et al., 1989).

7.2. Transforming growth factors (TGFs)

TGFs have diverse effects on growth and differentiation in normal and neoplastic cell types (Lawrence, 1989). Most important in skeletal tissue are members of the TGF-b gene family which includes the activins, inhibins, mullerian inhibiting substance, bone morphogenetic proteins (BMPs), the drosophila decapentaplegic gene complex product (dpp), and products of the mammalian Vgr gene. At least three isoforms of TGF-b have been isolated in mammalian tissues (TGF-b 1,2,3). There is considerable sequence identity and shared biological effects between these isoforms (Burt & Paton, 1992). TGF-b is produced by several cell types, with bone matrix one of the most abundant sources of both TGF-b1 and TGF-b2 (Seyedin et al., 1985). Regulation of TGF-b, like that of many cytokines, occurs not only at a transcriptional or translational level; it is secreted and stored in a latent form that requires activation to become functional. Considerable evidence exists supporting a role for TGF-b in morphogenesis, in the regulation of endochondral ossification and in bone remodelling (Kimelman & Kirchner, 1987; Ruscette & Palladino, 1991; Carrington et al., 1988). High levels of TGF-b messenger RNA are expressed in the growth plate of fetal human long bones (Sandberg et al., 1988), and TGF-b has been immunolocalised at sites of endochondral ossification in the developing mouse skeleton (Heine et al., 1987). TGF-b induces chondrogenic activity in mesenchymal cells (Seyedin et al., 1985), and accumulates during endochondral ossification in the demineralised bone implant model (Carrington et al., 1988). In vitro studies have shown that TGF-b regulates the synthesis of collagen by growth plate chondrocytes; increasing the synthesis of type I relative to type II collagen (Rosen et al., 1988; Sandell et al., 1989); it may therefore control mineralisation by regulation of hypertrophic chondrocyte differentiation. The effects of TGF-b on endochondral ossification may be to stimulate growth in the undifferentiated cell, with different effects on the terminally differentiated chondrocyte.

TGF-b has a role to play in regulation of bone remodelling, having effects on the proliferation and differentiation of osteoblastic cells in vitro (Bonewald & Mondy, 1990), and periosteal injection will stimulate bone formation in vivo (Mackie & Trechsel, 1990). TGF-b inhibits interleukin-1 and 1,25(OH)₂D₃ induced bone resorption and the formation of multinucleated osteoclast-like cells in a human marrow culture system (Chenu et al., 1988; Pfeilschifter, Seyedin & Mundy, 1988). These diverse effects of TGF-b on bone cells have led to the hypothesis that TGF-b may have a role in the coupling of bone formation to bone resorption. One proposed mechanism is that during bone resorption latent TBF-b is released from bone matrix and activated (possibly by the low pH and/or proteases), to act locally on bone cells (Bonewald & Mundy, 1990).

7.3. Bone morphogenetic proteins (BMPs)

This large family of proteins has aroused considerable interest in the bone cell field, since the discovery that the implantation of demineralised matrix at subcutaneous or intramuscular sites leads to bone formation (Urist, 1965). The factors in bone matrix responsible for this induction of bone formation were named the bone morphogenetic proteins (BMPs). There are now known to be 7 members of this family (BMPs 1-7); all except BMP1 are members of the TGF-b family. BMP1 has been classed as a novel regulatory protein (Wozney, 1989). Chromosome mapping has shown that the BMP2A and BMP3 genes map to conserved regions between mouse and human, while the BMP1 gene does not (Tabas et al., 1991). The term 'bone morphogenetic' may, however, prove to be a misnomer, since the messenger RNA for the BMPs are expressed in a wide variety of tissues (Bentley et al., 1992), suggesting limited tissue specificity of function.

BMPs are the only molecules so far discovered capable of independently inducing endochondral ossification in vivo. TGF-b1 and TGF-b2 enhance the osteoinductive properties of BMPs; however, injection of TGF-bs on their own leads to extensive fibrous tissue formation only (Bentz, Armstrong & Seyedin, 1987). The mechanism of action of the BMPs has yet to be defined. However, the availability of recombinant forms has led to much work on their biological activity in vivo and in vitro. Recombinant forms of BMP2 and BMP4 induce ectopic bone formation, and BMP2 will heal cortical bone defects by an endochondral process (Hammonds et al., 1991; Wang, Rosen & Cordes, 1990; Yasko et al., 1992). BMP2 stimulates the growth and differentiation of growth plate chondrocytes in vitro, and results in the development of the osteoblast phenotype in a rat pluripotential cell line (Hiraki et al., 1991). Osteoblasts have been shown to have high affinity binding proteins for BMP on the cell surface (Paralkar, Hammonds & Reddi, 1991).

Indirect lines of evidence demonstrate that BMPs have a critical role in bone development. Firstly, the protein encoded by the decapentaplegic locus (dpp) in Drosophila is a member of the TGF-b family member with 75% sequence homology to BMP2, suggesting a common ancestral gene. Developmental anomalies produced by mutations of the dpp gene are similar to patterns of disease expression in fibrodysplasia ossificans progressive, a developmental disorder characterised by deformations of the hands and feet and heterotopic chondrogenesis (Kaplan, Tabas & Zasloff, 1990). In addition, the chromosomal locations of the BMP genes overlap with the loci for several disorders of cartilage and bone formation (Tabas et al., 1991). More direct evidence is provided by a recent study which demonstrated that BMP2, together with fibroblast growth factor-4, is important in regulating limb growth in the mouse embryo (Niswander & Martin, 1993).

7.4. Fibroblast growth factor (FGF)

FGF is a heparin binding peptide that exists in two forms, acidic and basic, with 55% sequence homology between the two (Gimenez-Gallego, 1985). The regulation of the synthesis and secretion of FGF is not well understood, although the biological effects have been widely characterised. FGFs are potent mitogens for osteoblasts, chondrocytes and endothelial cells, and stimulate proliferation of mesenchymal cells in the developing limb that leads to limb outgrowth (Niswander & Martin, 1993). FGF receptors are expressed in limb mesenchyme as is mRNA for FGF-4 (Niswander & Martin, 1992; Suzuki et al., 1992).

There is increasing evidence that basic FGF (bFGF) is also important at later stages of bone growth, bFGF interacts with two classes of binding sites on bovine growth plate chondrocytes: a high-affinity bFGF receptor and a low-affinity heparin-like binding site (Tripper et al., 1992). FGF and IGF-1 are the minimum growth factor requirements for serum-free growth of avian growth plate chondrocytes (Leach & Rosselot, 1992), and bFGF increases collagen and proteoglycan synthesis by growth plate chondrocytes cultured from sheep, having a synergistic effect with TGF-b (Hill et al., 1992). FGFs are not secreted proteins since a leader sequence is lacking, so they may only be released from their cell of origin after membrane disruption. In this way FGF released from the degenerating chondrocyte may act as a mitogen for metaphyseal vessels (since FGF is a potent angiogenic factor) and cells of the osteoclast lineage. Like the growth factors discussed previously, bFGF is found in bone matrix, and has been shown to be synthesised by bone cells in vitro (Hauschka et al., 1986; Canalis, Centrella & McCarthy, 1988). During bone remodelling, FGF synthesised by osteoblasts and stored in bone matrix may be released following osteoclastic bone resorption. Activated FGF may then be important in stimulating bone formation by increasing the number of osteoblastic precursor cells. bFGF has no effect on osteoblast differentiation (Canalis, Centrella & McCarthy, 1988).

7.5. Platelet-derived growth factor (PDGF)

PDGF, a dimeric 30kDa peptide, was initially isolated from human platelets and is known to exist in both homo- and heterodimeric forms (Ross, 1989). PDGF has been found in bone matrix extracts and is secreted by human osteosarcoma cells and untransformed rat osteoblasts (Betsholtz et al., 1986). However, its synthesis by normal human osteoblasts or chondrocytes has not been reported. The PDGF located in bone matrix may be sequestered from the systemic circulation. PDGF is mitogenic for osteoblasts, fibroblasts and periosteal cells, although it is possible some of these effects may be mediated by IGF-I, since PDGF increases IGF-I synthesis in mesenchymal cells (Mohan & Baylink, 1991).

PDGF may play a role in bone development and growth, although little is known of the precise mechanism. PDGF gene expression has been reported in pre-implantation mouse embryos (Rappolee et al., 1988), but PDGF has no effect on limb bud elongation in mice (Niswander & Martin, 1993). Both homodimeric forms of PDGF bind and increase DNA synthesis in growth plate chondrocytes, having an additive effect with IGF-1 (Wroblewski & Edwall, 1991). This study also demonstrated an increase in alkaline phosphatase activity and c-fos expression in chondrocytes following PDGF stimulation.

Several other cytokines are known to be important in the regulation of bone and cartilage cells, although little is currently known of their role in normal endochondral ossification. In the stunted child, where disease may be a significant contributing factor, cytokine effects on the bone growth plate may be of particular importance. For example, in post-menopausal osteoporosis, cytokine production by circulating cells may be altered, and this mechanism is believed to be important in the uncoupling of bone formation from resorption characteristic of this disease (Pacifici et al., 1987).

7.6. Tumour necrosis factors (TNF)

Alpha and beta forms of TNF exist and, although there is only 28% sequence identity, they share the same receptors, and their range of biological activities overlaps, with many similar functions to IL-1. A second form of the TNF receptor exists that binds to circulating TNF, and is shed after cleavage of the extracellular TNF cell surface receptor. TNFa is produced by most cell types, including osteoblasts, in response to a range of non-specific signals (Gowen et al., 1990). TNFb is only induced by specific antigens and has only been shown to be synthesized by activated T cells (Beutler & Cerami, 1988). In skeletal tissues, TNFs stimulate bone and cartilage resorption and cell division (Gowen, MacDonald & Russell, 1988; gunning & Russell, 1989). TNFa has been shown to reversibly inhibit ectopic bone formation in mice by preventing mesenchymal cells from differentiating into chondrocytes (Yoshikawa et al., 1988). Since TNFa induces neo-vascularisation in vivo, it may work with other local factors, including FGF and TGFa to stimulate vascular invasion of the growth plate (Folkman & Klagsbrun, 1987).

7.7. Interleukin 1 (IL-1)

IL-1 exists in two 17 kDa forms, alpha and beta, that have a similar spectrum of biological activity but little sequence homology. IL-1 was originally isolated from cells of the monocyte series but has subsequently been shown to be expressed by most cell types, including human osteoblasts (Keeling, Rifas & Harris, 1991). The range of biological effects of IL-1 is extensive, with activities previously attributed to leucocyte endogenous mediator (LEM), mononuclear cell factor (MCF), osteoclast activating factor (OAF) and catabolin now known to be those of IL-1 (Dinarello, 1987). The first cell surface receptor to be identified for IL-1 was found to be a member of the immunoglobulin superfamily (Sims et al., 1988), and it is felt that cytokine interactions occur at this level of the receptor, with receptors for PDGF, IL-6 and the colony stimulating factors (CSFs) included in this family. A naturally occurring specific antagonist has been recently characterised that competes for occupancy of the cell surface receptor (IL-1ra, Dinarello & Thompson, 1991). There is also evidence that there may be a soluble form of IL-1 receptor (Symons, East-gate & Duff, 1991). The most potent inducer of IL-1 synthesis is endotoxin, but it is also induced by a number of other cytokines and in an autocrine manner by IL-1. IL-1b stimulates bone resorption in vitro and in vivo, and increases the proliferation of osteoblast cells and the production of other cytokines by osteoblasts (Gowen et al., 1983; Gowen, Wood & Russell, 1985; Gowen et al., 1990). IL-1 mRNA has been localised in the calcified cartilage zone of growth plate, and together with BMP enhances ectopic bone formation in vivo, and cartilage formation in vitro (Mahy & Urist, 1988). Since IL-1 suppresses cell proliferation and proteoglycan synthesis in chondrocytes, and decreases types II and IX collagen synthesis, it may suppress the cartilage phenotype in the hypertrophic zone that precedes the onset of mineralisation (Ikebe, Hirata & Koga, 1988; Goldring et al., 1988). Local synthesis of IL-1 and TNFa may also be important in the remodelling of matrix in the metaphysis via stimulation of the synthesis of proteinase enzymes by bone and cartilage cells.

7.8. Interleukin 6 (IL-6)

IL-6 is a 23-28 kDa protein produced by many cell types including fibroblasts, bone and cartilage cells as well as monocytes. Synthesis in osteoblastic cells is stimulated by a range of factors including IL-1 and PTH (Linkhart et al., 1991; Lowik et al., 1989; Feyen et al., 1989). The considerable overlap in the biological activities of IL-6 and IL-1 has led to the suggestion that IL-6 mediates some of the actions of IL-1. The role that IL-6 plays in bone is not clear. Direct effects have been demonstrated in osteosarcoma cells, although it has not been shown to affect cell growth or differentiation in primary cultures of human osteoblast cells (Fang & Hahn, 1991; Littlewood et al., 1991). Similarly the effect of IL-6 on bone resorption is not well defined. In two studies involving the mouse calvarial system IL-6 had no effect on resorption, whereas others reported that IL-6 stimulated resorption (Al-Humidan et al., 1991; Ishimi, Miyaura & Jin, 1990). There is also evidence that IL-6 may mediate some of the effects of oestrogen on bone.

7.9. Interleukin 8 (IL-8)

IL-8, or neutrophil activating factor (NAF), is an inflammatory mediator produced by a wide variety of cell types. IL-8 is a potent attractant for neutrophils and may have an important role to play in diseases such as rheumatoid arthritis and osteoarthritis (Peveri et al., 1988). Other members of the IL-8 supergene family may also have effects within connective tissues, including the monocyte chemotactic and activating factor (MCAF), macrophage inflammatory protein (MIP-2) and platelet factor-4.

7.10. Interferons (IFN)

This is a family of molecules that are potent inhibitors of malignant and normal cell proliferation. There are three types, alpha, beta and gamma, and of these only IFNg has significant osteotropic effects. Its principal role is believed to be that of an antagonist to IL-1 and TNFa induced bone resorption. IFNg inhibits bone resorption, in part, by inhibiting osteoclast formation from precursors (Hoffmann et al., 1987; Takahashi, Mundy & Roodman, 1986).

7.11. Colony stimulating factors (CSFs)

These molecules are of importance in haematopoetic differentiation and those studied most in relation to bone are the monocyte./macrophage CSF (M-CSF), granulocyte-macrophage (GM-CSF) and multi-CSF (IL-3), because of the assumption that osteoclasts and monocytes share a common ancestor. Both GM-CSF and M-CSF are produced by marrow stromal cells, and M-CSF is known to be required for normal osteoclast development (Felix, Elford & Stoerckle, 1988). In the 'op/op mouse', a naturally occurring model in which the M-CSF gene is mutated, the animals have osteopetrosis (a condition of impaired bone resorption), and this can be reversed by the injection of M-CSF (Felix, Cecchini & Fleisch, 1990).

7.12. Parathyroid hormone related peptide (PTHrP)

PTHrP is a peptide closely related to PTH that is produced by normal tissues, with similar effects to PTH on bone. It has been established as having an important role in regulating the hypercalcaemia that is associated with some malignancies (Webb et al., 1988). PTHrP has also been identified as a fetal hormone which may regulate placental calcium (Ca²⁺) flux (Orloff, 1989). This peptide may also have an important role in skeletal development, having been localised in embryonic bone, and a recent study has shown that mice with a defective PTHrP gene have multiple skeletal abnormalities (Karaplis et al., 1992).

7.13. Calcitonin gene related peptide (CGRP)

This peptide is a separate product of the calcitonin gene. There is increasing evidence to suggest it is important in the local regulation of skeletal tissues. CGRP inhibits bone resorption (De Souza et al., 1986), it has effects on osteoblast cells, and may regulate cytokine synthesis by osteoblast precursors (Michelangeli et al., 1989). Its location in nerve cells is evidence for a potential role as a neurogenic modulator of bone cells (Weigent & Blalock, 1987).

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