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8. Regulation and mechanisms of cytokine action

Cytokines have overlapping activities despite structural differences. Each cytokine may have a wide range of biological effects, implying that several regulatory mechanisms exist for specificity of action to be preserved. Levels of biologically active cytokines may be regulated by changes in gene transcription, mRNA stability and translational and post-translational modifications. Several cytokines are synthesised as an inactive precursor protein that requires subsequent cleavage, e.g. IL-1, TGFb and IGF-II (Dinarello, 1987; Derynck, 1985; Bell et al., 1984). Another regulatory mechanism exists in hard tissues where matrix acts as a reservoir for several growth factors. Availability of cytokines to cellular receptors may also be regulated by binding to specific binding proteins (BPs) in serum and tissue. Some of the best characterised BPs to date are the IGF binding proteins (IGFBPs). Cytokine receptors are found in several cell types throughout the body, and specificity of response may be exerted through alterations in receptor numbers and their binding affinity for respective ligands. Inhibition of cytokine activity may also occur at the level of the cell surface receptor. A specific inhibitor has recently been cloned that binds to the IL-1 receptor but lacks agonist activities, the IL-1 receptor antagonist (IL-1ra) (Dinarello & Thompson, 1991). IL-1b may be further regulated via a binding protein present in human plasma that may be a soluble form of the IL-1 receptor (Symons, Eastgate & Duff, 1991). Receptors shed from the cell surface may bind and neutralise cytokine activity. Two forms of the TNFa receptor act in this manner (Beutler & Cerami, 1988). A number of cytokines, including IL-1 and TNF, can regulate the number of their own receptors or modulate receptors of other cytokines (Mizel et al., 1987). These observations, and the demonstration that homologues share the same receptor, help to explain the synergistic actions of cytokines.

Cytokine-receptor binding induces cellular responses via signal transduction pathways. This may be via the release of second messengers such as cAMP, Ca2+, and phosphoinositols, and there is an important process of phosphorylation or dephosphorylation of specific proteins. Changes in the receptor after ligand binding may also be important. The receptors for PDGF, IGF-1 and EGF have intracellular regions with intrinsic tyrosine kinase activity, an enzyme important in regulating protein phosphorylation, that becomes activated after the cytokine binds the receptor.

The final response of the cell is a modulation of the activity of certain target genes, and this depends partly on the modification of DNA binding proteins, the transcription factors. Some of these protein-DNA interactions are modified by phosphorylation or dephosphorylation of the protein elements. One class of transcription factors that control the transcription of a variety of genes are the products of oncogenes, which are known to have important roles in cell proliferation and differentiation (Johnson & McNight, 1989). There is evidence that growth factors may act through induction of oncogenes. fos and jun mRNAs are induced in cultured cells within a few minutes of exposure to exogenous growth factors or serum (a rich source of growth factors and hormones) (Barter et al., 1989). The oncogene products of jun and fos (AP-1), form a heterodimer which binds to DNA at the region of a gene promoter to form the transcription factor complex. A number of cytokines have been shown to increase expression of c-jun and c-fos (Brenner et al., 1989; Pertovaara et al., 1989), and other growth factors such as PDGF, acting through the tyrosine kinase cascade, may modify phosphorylation of the AP-1 heterodimer and subsequent DNA binding.

The role of proto-oncogenes in regulating genes, and their induction by local factors, implies that they have effects in a range of tissues, including bone and cartilage. AP-1 activity is increased in the osteocalcin gene promoter region during osteoblast proliferation, whereas fos and jun expression are reduced when the cells have stopped dividing and differentiation is increased (Owen et al., 1990). c-fos mRNAs are expressed in embryonic human long bones (Sandberg et al., 1988), and another oncogene product, the c-myc, is actively synthesised by both proliferating and differentiated rat and chick growth plate chondrocytes (Farquarson, Hesketh & Loveridge, 1992). More direct evidence for oncogenes being important in skeletal development is provided by a recent study in which transgenic mice were created in which the c-fos gene was disrupted. In homozygous mice, growth, epiphyseal and metaphyseal enlargement were retarded and the growth plate was highly disorganised. Cortical bone was poorly developed, osteoclast numbers were reduced and there was evidence of osteopetrosis (Wang et al., 1992). The development of transgenic models has potential for the field of cartilage and bone cell biology as it enables the specific effects of a hormone/cytokine/oncogene on skeletal development to be investigated. Notwithstanding, genetic manipulation cannot take into account the redundancy that exists in nature, such that genes within closely related families may share or take over each other's function. This is believed to explain why disruption of the TGF-b gene, closely related to several developmentally important genes including the BMPs, produces no skeletal abnormalities in the mouse (Shull et al., 1992).


The field of bone cell biology is clearly of relevance to the problem of stunting in children, as in the final analysis the cells of the growing long bone are the ultimate 'regulators'. It is the alterations in the functions of these cells that manifests as a reduction in height. Normal longitudinal growth is achieved by the coordinated recruitment, proliferation, differentiation, maturation and eventual death of the cells of growth plate and bone. Cellular activity is closely regulated by endocrine factors acting directly or indirectly, with factors produced locally and stored within the bone and cartilage microenvironment having a critical role in intercellular communication. Disruption of any of these processes can lead to growth disturbances, since it only requires a defect in a single gene to have profound effects. Studies in recent years have shed light on the biochemical and molecular effects of cytokines and growth factors and have shown that these regulatory molecules may mediate the effects of certain hormones important in controlling growth. However, the complex interrelationship of these molecules is still not clear. Notwithstanding, understanding of the mechanisms involved in bone remodelling is increasing, as this area attracts much research because of the high incidence of metabolic bone disease in Western society.

Although studies of adult bone remodelling are of relevance, there is a requirement for increased research directed specifically at the mechanisms of endochondral ossification and its regulation. Longitudinal bone growth is a challenge to the cell biologist, since it is an accelerated cycle of cellular division and differentiation, within which it is not easy to separate events temporally and spatially. In addition, different regulatory mechanisms are probably important at different stages of growth. Another difficulty impeding progress in this field is the lack of appropriate animal models for research. Much information has come from studies involving rodents, and species differences must always be taken into account. Larger mammals such as the growing piglet or the calf are probably more appropriate for the study of postnatal longitudinal growth in man.

If the mechanisms of stunting are to be established at a cellular level, a number of approaches need to be considered. Studies need to be designed using more appropriate animal models, and conditions such as nutritional intake, immunological challenges, chronic intestinal diseases and mechanical loading need to be manipulated. Any effects on longitudinal growth may then be studied temporally and correlated with non-invasive measurements including assays of hormones, cytokines, growth factors and proteins known to regulate their activity. Alterations in growth plate structure and cellular function can be studied in these models in situ using a variety of histological, biochemical and molecular approaches. In vitro studies using cultured growth plate cells, preferably of human origin, are also required for the study of how hormones and cytokines transduce their signals intracellularly.

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