| dc.description.abstract | A number of growth factors and cytokines involved in the local regulation of bone
remodelling are either synthesised by osteoblasts or have osteoblasts as their target.
These include the RANK-L/OPG system, the gp130 cytokine family, including IL-6, and
insulin like growth factors. In addition, aberrant cytokine signalling is strongly linked
with pathological states characterised by increased bone resorption, including
osteoporosis and renal osteodystrophy. The range of action and potency of these
osteotropic cytokines requires that their actions are tightly regulated. Amongst such
potential control mechanisms are the suppressors of cytokine signalling (SOCS), the
presence and role of which in bone has not been studied in detail.
The aim of this thesis was (i) to examine the direct effect of uraemia on cytokine
release in human osteoblastic cells; (ii) to determine if the regulatory SOCS genes are
expressed in these cells and, if so, (iii) to characterise their functional significance.
In initial studies, osteoblastic cells were cultured in media containing sera from either
healthy volunteers or haemodialysis treated chronic kidney disease patients.
Concentrations of OPG and IL-6 were then measured in harvested supernatants.
Additionally, individual serum samples collected prior to, and during, a haemodialysis
(HD) session were assayed for IL-6, IL-1β and soluble IL-6 receptor (sIL-6R). HD
patients had significantly higher concentrations of IL-6 than normal subjects, but there
were no significant differences in either IL-1β or sIL-6R. These concentrations did not
change significantly during HD. There were no differences in OPG production by
osteoblastic cells after exposure to either normal or uraemic serum. Incubation with
untreated sera from normal subjects increased IL-6 production by ~6-fold above
control, whereas sera from uraemic subjects increased it only ~2-3-fold. HD did not
restore the capacity of uraemic serum to augment IL-6 release to the same degree as
normal serum.
Further work examined a variety of osteotropic stimuli for their ability to induce SOCS1-
3 and CIS expression in human osteoblastic cells. The utility of both conventional RTPCR
and fluorescence-based kinetic real time PCR for this purpose are compared.
These SOCS were found to be expressed constitutively and could be induced to a
variable degree by relevant growth factors. In general, the temporal pattern of SOCS
expression was consistent with a negative feedback function. Potential functionality
was explored following transfection with SOCS1 and SOCS3 plasmid DNA.
Significantly enhanced IL-6 secretion was found in both the basal and stimulated state,
whilst OPG production was enhanced only in the latter. Function was also studied in
the context of osteoblastic apoptosis, the regulation of which is highly relevant to
skeletal disease. Initial experiments developed a framework for subsequent studies:
serum starvation for 24h produced reproducible cell death that could be attenuated in a
dose dependent manner by IGF-I. SOCS1 and SOCS3 overexpression had limited
influence on osteoblast survival, whereas gene knock down experiments using siRNA
indicated that IL-1β-induced cell death is mediated differentially, depending on the type
of cell death involved. SOCS1 and SOCS3 are involved in the apoptotic cascade, while
IL-1β-induced necrosis appears to be independent of SOCS3.
Collectively these studies demonstrate that the augmentation of IL-6 production by
osteoblastic cells after exposure to normal serum is greater than after uraemic serum.
HD does not correct this disparity; perhaps indicating a non-dialysable inhibitor of IL-6
release is involved in the dysregulated bone turnover of uraemic patients. Further work
establishes the constitutive presence of the SOCS family in human osteoblastic cells,
as well as their transient inducibility by key osteotropic stimuli. Several novel aspects of
SOCS function, including influence on IL-6 and OPG production and involvement within
apoptotic pathways are demonstrated. | en_US |
