SKIN & BONE LABORATORY

(ENDOCRINE REGULATION)

Rebecca S. Mason

  

  

RESEARCH

PERSONNEL in 1994 and 1995

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)
1993-
Belinda Welsh MMed student (Supervisor:
G. Halliday, Dept of Dermatology)
Dermatology
Foundation S'ship
1995-
Omar Chani GradDipSc student (Supervisor:
Chris O'Neill, Obstet. & Gyn., RNSH)
1995-

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.
  

PROJECTS in 1994

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.

Oncogenic osteomalacia

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 PLANNED for 1995

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.
  

COLLABORATIONS

Production and function of parathyroid hormone-related peptide in skin:
Dr Jane Moseley, St Vincent's Institute for Medical Research, Melbourne (1990-present) and Ms Margaret Wilkinson, Endocrine Laboratory, Royal North Shore Hospital (1993-present).

Adaptive responses to solar-simulated irradiation:
Gavin Greenoak, Dept of Animal Science (1991-present) and Dr Rakesh Kumar, Dept of Pathology, Univ. of N.S.W. (1992-present).

Hormonal modulation of bone-like formation in vitro and influence of vitamin D receptor alleles:
Dr Bernie Tuch, Dept of Endocrinology, Prince of Wales Hospital; Dr Brian Luttrell and Margaret Wilkinson, Endocrine Laboratory, Royal North Shore Hospital (1990-present); and Prof. John A. Eisman and Dr Nigel Morrison, Garvan Institute, St Vincent's Hospital (1993-present).

Pharmacological glucocorticoids and bone:
Prof. Ken Brown, Dept of Pharmacy; and Prof J. Paul Seale, Dept of Pharmacology (1994-present).
  

FACILITIES

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.
  

THESES PASSED in 1994

PhD

McLeod S (1994) Hormonal modulation of human melanogenesis.
  

SCHOLARSHIPS in 1994

Faculty of Medicine Postgraduate Scholarship, M. Slater, 1992-4.
  

OTHER RESEARCH ACTIVITIES in 1994

Refereeing

Manuscripts: for Clinical Chemistry (1).

Grant applications: for NHMRC (3), Anti Cancer Foundation, S.A. (1), Ramaciotti Foundation (1), Community Health & Anti-Tuberculosis Assn (1).

MMed thesis examined: for Univ. of Sydney (1).

PhD thesis examined: for Univ. of Queensland (1), Univ. of Adelaide (1), Univ. of W.A. (2).

Official of scientific societies

Member, Programme Committee for Annual Scientific Meetings of Australian and New Zealand Bone and Mineral Society (1991-94).

Member, local organizing committee for XIIth International Conference on Calcium Regulating Hormones, to be held in Melbourne in 1995.

Member, Programme Committee, Endocrine Society of Australia Seminar Meeting (1993-5).

Local seminars

Dept of Medical Oncology, Westmead Hospital (May).

Dept of Endocrinology, Royal Prince Alfred Hospital (Aug).

Dept of Pharmacology (Oct).
  

5-YEAR RESEARCH PUBLICATIONS

MOST RECENT TOTAL ANNUAL CITATIONS (for 1993): 23

  

FUNDING in 1994 and 1995

NHMRC Direct and indirect effects of estrogens
in a human bone formation model
Mason RS 1992
1993
1994 $45,155

NHMRC Influence of vitamin D receptor polymorphism
on osteogenic activity in culture
Mason RS 1995 $45,029
1996
1997

NSWSCC Epidermal cell-derived cytokines and the adaptive
responses to UV irradiation
Mason RS 1994 $33,341
1995 $63,901
1996

USCRF Molecular mechanisms of the phosphate
defect in oncogenic osteomalacia
Mason RS 1995 $24,000

Total for 1994: $78,496

Total for 1995: $132,120

  

  

TEACHING

COURSES TAUGHT

Medical Science 2: Human Life Sciences 2

Lectures: 4, on endocrinology of pancreas, thyroid and calcium regulation.

Tutorials: 1 on endocrine physiology.

Tests: Multiple choice questions provided for informal assessment.

Assessment: Semester exams by short answer questions; 1 essay during semester.

Medical Science 3: Human Life Sciences 3

Lectures: 2 on chemical signalling.

Medical Science 3/Science 3: Cardiovascular

Lectures: 2 on lipid metabolism and development of atheroma.

Essays: 1 on cardiovascular, to a fraction of students, completed during term.

Examination: Essay-style question.

Dentistry 2/Science 2/Science 2 Auxiliary

Lectures: 2 on mineral metabolism.

Tutorials: 1, presented twice, with quiz.

Examination: Multiple choice questions (of the 5 alternatives, 1 correct, variety) and short answer questions.

Medicine 2

Lectures: 18: 4 on endocrinology of pancreas, thyroid and calcium regulation, 14 on respiratory physiology.

Practical classes: 1, of 3 h, presented 8 times, on endocrine physiology, including a new practical session based on an introduction to clinical skills relating to hypo- or hyperthyroidism. Office laboratory tests for blood glucose concentrations or pregnancy were shown.

1, of 3 h, presented 8 times, on respiratory physiology, including a new clinical skills segment and office testing of lung function.

Tutorials: 1 h follow-up session presented 8 times, involving student presentation of results and tutorial on endocrine physiology.

1.5 h follow-up session, presented 4 times, involving a respiratory physiology video, with question and answer session and general presentation and discussion of results of respiratory practical.

Examination: Multiple choice and short answer questions.

Course development: 2 new segments on basic clinical skills introduced to practical classes were well received by students.

Medicine 3 (Clinical Physiology with Pharmacology)

Course Supervisor

Lectures: 4, on clinically-applied endocrine physiology.

Examination: Multiple choice and essay questions.

Pharmacy 1

Lectures: 10: 3 on endocrinology of pancreas, thyroid and calcium regulation, and 7 on respiratory physiology.

Tutorials; 1, including some example multiple choice questions.

Examination: Multiple choice questions (of the 5 alternatives, 1 correct variety).
  

TEACHING HOURS in 1994

Dent2/
Sc2/ScAux
Med 2 Med 3 Pharm1 HLS 2 HLS3/
Sc3
Total
Lectures 2 18 4 10 4 4 42
Practical classes (no.) - 48 (16) - - - - 48
Tutorials 2 14 - 1 1 - 18

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.
  

  

ADMINISTRATION

(see also OTHER RESEARCH ACTIVITIES in 1994)

University committees

Member, Special Studies Program Committee (1993-94).

Academic Board

Elected Faculty member (non-professorial) (1994-).

Member, Committee for Graduate Studies (1993-).

Member, Committee for Undergraduate Studies (1994-).

Nominee of Academic Board for Appointment Committees.

Faculty of Medicine committees

Sub-dean, Staff-student Liaison Committee.

Member, Teaching Management & Assessment Committee (Undergraduate Medical Degree).

Member, Dean's Planning and Advisory Committee.

Member, Year 3 Curriculum Committee.

Member, Basic and Clinical Sciences Committee, Graduate Medical Degree.

Co-coordinator, Endocrinology and Metabolism Planning Team.

Member, Core Promotion Committee (Lecturer to Senior Lecturer) with associated Clinical Titles committees.

Member, Medical Library Advisory Committee.

Member, Committee on Restriction on Re-enrolments

Member, Bancroft Prize Committee.

Department of Physiology

Secretary, Departmental Board.

Community and hospital

Member, Quality Control Committee, Endocrine Laboratory, Royal North Shore Hospital.