|Dr Rebecca S. Mason||Senior Lecturer (in-charge)||University||1988-|
|Nalini Dissanayake||PhD student (from 91)||NSWSCC||1989-|
|Stephen D. McLeod||PhD student (part-time)||USCRF Grant S'rship||1990-94|
|Michael Slater||PhD student (from 92)||Fac. Med. S'ship||1991-95|
|John Patava||PhD student (from 92)||NHMRC Grant S'ship||1991-|
|Heeja Namkung||PhD student (from 93)||overseas student||1992-|
|Anne Nelson||PhD student (Assoc. Supervisor:
B. Robinson, Dept of Medicine)
|Belinda Welsh||MMed student (Supervisor:
G. Halliday, Dept of Dermatology)
|Omar Chani||GradDipSc student (Supervisor:
Chris O'Neill, Obstet. & Gyn., RNSH)
Current effective full-time personnel (co-supervised as 0.5) = 5.0
Research in this Laboratory examines how skin cell function and bone forming activity are regulated, with special emphasis on the roles of hormones and cytokines in these processes.
Hormonal control of pigmentation
Steve McLeod extended previous studies on the mechanism of increased pigmentary activity due to pro-opiomelanocortin derived peptides. The studies showed that adrenocorticotrophic hormone (ACTH) was the most potent natural modulator of pigmentation amongst this group of peptides. As seen in studies on adaptive responses to UV irradiation (see below), there is strong evidence that skin cell-derived ACTH participates in the pigmentary response after UV irradiation. Steve used a variety of stimulators and relatively specific inhibitors of protein kinase C and cAMP-dependent protein kinase (PKA), as well as direct measurement of kinase activity, to obtain data consistent with our hypothesis that increased pigmentary activity after ACTH is mainly modulated by the protein kinase C pathway (despite ACTH being well known to increase cAMP production), with the PKA pathway playing a facilitative role. The studies also provided an explanation for the empirical observation that cultures of melanocytes must be allowed to recover from chronic stimulation with phorbol ester, which down regulates PKC activity, before a response to ACTH can be detected.
Adaptive responses to UV irradiation
The studies, by Nalini Dissanayake, concentrated on the role of parathyroid hormone related peptide (PTHrP) in these responses. In the skin cell culture model, UV irradiation induces a late increase in proliferation of epidermal cells as well as an increased cornification response (equivalent to the increased barrier layer in UV exposed skin) and increased pigmentary activity of melanocytes (equivalent to a tan). These adaptive responses have been linked in earlier work in this lab to release of ACTH (pigmentation) and transforming growth factor-beta (TGF-b)/PTHrP for the cornification response. A UV-dose-response and time course for PTHrP release after UV were determined. The capacity of TGF-b to stimulate PTHrP release was established. Increased PTHrP mRNA was detected in UV irradiated epidermal cells by in situ hybridization (in collaboration with Dr Jane Moseley, St Vincents Institute of Medical Research, Melbourne). Data suggesting potential, though modest, involvement of released TGF-b and PTHrP in the pigmentary response was also obtained.
Modulation of in vitro bone-like formation
For some years, there has been considerable controversy in the bone field concerning whether the active vitamin D metabolite, 1,25 dihydroxyvitamin D3 (1,25D3) facilitates mineralization of bone matrix simply by allowing an adequate supply of calcium and phosphate from the diet, or whether it has an additional direct effect on the deposition of mineral. The human cell based bone formation model, developed in the laboratory by John Patava, in which bone-derived cells in long term culture form a relatively orderly bone-like matrix which mineralizes, provided an opportunity to study the role of 1,25D3 in a system with fixed concentrations of calcium and phosphate. The studies showed mineral deposition only near the lower surface of the multilayer, consistent with 'physiological' mineral deposition. Using immunohistochemistry and biochemical studies of radiolabelled calcium deposition and a new technique of radiolabelled tetracycline deposition, with appropriate controls, John obtained evidence consistent with the hypothesis that the presence of 1,25D3 does indeed enhance mineralization in the presence of fixed concentrations of calcium and phosphate Complementary studies by Michael Slater showed that marked changes in the matrix accumulation of alkaline phosphatase and osteocalcin, both proteins believed to be involved in mineralization, occurred in the presence of 1,25D3 in a time- and dose-related manner. Michael also found evidence that the matrix protein thrombospondin was modulated by estradiol treatment in a similar manner to the growth factor TGF-b and co-localized with TGF-b in the matrix. This observation, combined with other studies in non-bone tissues, are consistent with the proposal that thrombospondin may act as a modulator of growth factor activity or as a growth factor-like agent in bone.
Glucocorticoids and bone
Long term exposure to high doses of glucocorticoids is known to result in reduced bone mass. There are several potential mechanisms for this, not all directly bone related. Although glucocorticoids tend to inhibit bone cell proliferation and activity in a number of rodent cell and organ culture models, there is little direct evidence for inhibitory effects of glucocorticoids on human bone cell (osteoblast) activity. One of the few papers on the subject suggested that glucocorticoids may have a role in facilitating differentiation of osteoblast precursors. Heeja Namkung carried out several studies on bone cell responses to the glucocorticoid dexamethasone using the bone cell system described above. Her work showed that while indeed dexamethasone was an inhibitor of bone-derived cell (osteoblast precursor) proliferation, it markedly stimulated differentiated functions such as alkaline phosphatase activity and protein synthesis in a dose- and time-dependent manner. Inhibitory effects of glucocorticoids on bone mass may well be a result of reduced numbers of bone forming cells, though not apparently reduced activity of these cells, especially under conditions where bone turnover is high due to effects of glucocorticoids on resorbing cell activity and inhibitory effects on gut and renal calcium conservation.
Studies by Ann Nelson showed that cells grown from a tumour that produced the syndrome of low blood phosphate and impaired bone mineralization in vivo (oncogenic osteomalacia) secreted into their culture medium, material capable of inhibiting phosphate uptake into opossum kidney (OK) cells. The material was as effective as authentic parathyroid hormone in this respect, although it differed from PTH in a number of other ways. RNA from cells actively secreting this material was extracted and used to construct a library (with commercial assistance). The process of constructing appropriate probes to screen this library is now underway. Probes to examine the molecular mechanism of the phosphate uptake inhibitory activity have also been prepared.
Research on the adaptive responses to UV irradiation will highlight the role of PTHrP and examine the molecular mechanisms of action of the skin-derived cytokines. The possibility that genetic variation in terms of vitamin D receptor alleles may determine, in part, responses of bone-derived cells to 1,25D3 or sex steroids will be studied. The glucocorticoid and bone studies will be extended to examine whether glucocorticoids modulate bone cell responses to local growth factors. In collaboration with Profs Ken Brown and J. Paul Seale, other glucocorticoids will be studied in order to determine their potency with respect to bone cell proliferation and function. Further studies to characterize the oncogenic osteomalacia derived-factor will continue at the biochemical and molecular level.
The Laboratory occupies room 237 of the Anderson Stuart Building. Dr Mason's office is room 245. The Laboratory is equipped for tissue culture studies and has a biohazard laminar flow facility, CO2-regulated incubator, tissue culture microscope and access to a Departmental liquid nitrogen storage facility. A second laminar flow hood houses a device for UV irradiation of cells. Equipment used for biochemical studies includes ultrasonic cell disruptor, shaking refrigerated water bath, pH meter, fluorimeter, gamma counter, balances and liquid scintillation counter. The laboratory also houses a Waters high performance liquid chromatography system with automatic injector, programmable gradient pump system, UVvisible detector, programmable fraction collector and chart recorder.
Faculty of Medicine Postgraduate Scholarship, M. Slater, 1992-4.
MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 23
|NHMRC||Direct and indirect effects of estrogens
in a human bone formation model
|NHMRC||Influence of vitamin D receptor polymorphism
on osteogenic activity in culture
|NSWSCC||Epidermal cell-derived cytokines and the adaptive
responses to UV irradiation
|USCRF||Molecular mechanisms of the phosphate
defect in oncogenic osteomalacia
Total for 1994: $78,496
Total for 1995: $132,120
|Med 2||Med 3||Pharm1||HLS 2||HLS3/
|Practical classes (no.)||-||48||(16)||-||-||-||-||48|
Total formal contact teaching time = 108 h
In addition, time was spent on lecture preparation, student consultations, exam setting, exam marking, essay marking, Honours thesis marking, course supervisor duties and curriculum development.
(see also OTHER RESEARCH ACTIVITIES in 1994)