SKIN & BONE LABORATORY

(ENDOCRINE REGULATION)

Rebecca S. Mason

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.

RESEARCH in 1993

Hormonal control of pigmentation

Steve McLeod extended previous studies in the Laboratory concerning the mechanism of development of melasma - the pigmentation often seen on the face of women who are pregnant or taking oral contraceptive medication. The work showed that incubation of melanocytes from a majority of donors in the presence of 17[[beta]]-estradiol resulted in an increase in melanogenic activity. Qualitatively similar results in the presence of the 17[[alpha]] analogue of estradiol and the failure of Tamoxifen or a 'pure' estrogen antagonist to inhibit this effect were inconsistent with the hypothesis that the 17[[beta]]-estradiol was acting as a classical steroid hormone. An alternative proposal, based on results using 2-hydroxy-estradiol, was that the estrogens were converted to catechol metabolites by enzymes shown to be present in other cells of neural crest origin (where this conversion works equally with [[alpha]] and [[beta]] analogues) and that direct interaction of these catechol intermediates with tyrosinase resulted in the observed effects.

Adaptive responses to UV-irradiation

These consist mainly of the increased depth of the cornified layer and increased melanin production and distribution, both of which confer some protection against subsequent radiation. Using an experimental model developed by Nalini Dissanayake, it was shown that directly irradiated skin cells display changes, in culture, consistent with those observed in vivo and that cytokines released by irradiated skin cells induce similar responses in cells which have never received irradiation. Nalini's studies provided some evidence to suggest that adreno- corticotropic hormone, or a similar peptide, mediates the increased melano- genesis after UV irradiation and that transforming growth factor-[[beta]] is involved in the cornification response, possibly in conjunction with another peptide - parathyroid hormone-related peptide. In collaboration with R. (first name?) Kumar, an epidermal growth factor-like compound with heparin-binding capacity, properties consistent with amphiregulin, was found to be released after UV irradiation and may be involved in the increased proliferation of epidermal cells noted under these conditions.

Modulation of in vitro bone-like formation

Long term cultures of human osteoblast-like cells, developed by John Patava, were used to examine growth factor incorporation into the matrix of cultured 'bone-like' material. It has been speculated that during bone turnover, these growth factors may be released during resorption and may act as 'coupling agents' to attract new osteoblast progenitors and direct their activity. John found that addition of 17[[beta]]- but not 17[[alpha]]-estradiol to the cultures stimulated forming activity, probably by increasing local growth factor production by the cells. A similar response was seen by Michael Slater when a source of growth factors, in the form of human platelets, was added to the cultures. Examination of the matrix of the cultures, using methods developed by Michael, which detect, by immunogold electron microscopy, either growth factor protein or growth factor mRNA, showed that whereas incorporation of transforming growth factor-[[beta]] and insulin-like growth factors I and II was increased in cultures treated with estradiol, this was not the case when growth factors were added exogenously. The results suggest that while estradiol increases local growth factor production, it has an additional effect to increase the incorporation of those growth factors into the matrix. The presence of adequate amounts of sex steroids (where a similar effect was seen with dihydrotestosterone) may thus enhance current bone forming activity and assist in the maintenance of bone after the next resorption cycle.

Phosphate transport in renal and bone cells

Evidence for the presence of a sodium-dependent phosphate transporter in normal cultured human bone cells, analagous to that found in renal cortical cells was obtained in studies by Heeja Namkung. Her experiments also suggested that phosphate transport in this system was regulated by parathyroid hormone, 1,25 dihydroxyvitamin D3 and glucocorticoids. Two probes to rat renal phosphate transporters were prepared by Ann Nelson and will be used to study phosphate transport in opossum kidney (OK) cells after treatment with extracts or supernatant from a tumour which caused oncogenic osteomalacia and, if there is cross hybridization, the bone cell transporter.

RESEARCH PLANNED for 1994

Research on the adaptive responses to UV irradiation will concentrate on the role of parathyroid hormone-related peptide in the cornification response and on studies using very low dose UV radiation. The potential capacity of 1,25 dihydroxyvitamin D3 to sensitize osteoblasts to estradiol or growth factor effects will be examined. The exciting possibility that the biological variability that was observed amongst osteoblasts from different donors, in terms of the magnitude of the responses to 1,25 dihydroxyvitamin D3, may be due, in part, to possession of different alleles of the vitamin D receptor, will be explored in collaboration with John Eisman's group at the Garvan Institute. Further characterization of the bone phosphate transporter will also be carried out.

PERSONNEL in 1993 and 1994

Rebecca S. Mason Senior Lecturer (in-charge) University 1988-

Nalini Dissanayake PhD student (from 1991) NSWSCC 1989-

Stephen D. McLeod PhD student (part-time) USCRF grant sch. 1990-

Michael Slater PhD student (from 1992) Fac Med sch'ship 1991-

John Patava PhD student (from 1992) NHMRC grant sch. 1991-

Heeja Namkung PhD student (from 1993) Overseas student 1992-

Ann Nelson PhD student 1993-

COLLABORATIONS

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

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

Hormonal modulation of bone-like formation in vitro and influence of vitamin D receptor alleles: Dr B. Tuch, Dept of Endocrinology, Prince of Wales Hospital; Dr B. Luttrell and M. (first name?) Wilkinson, Endocrine Laboratory, Royal North Shore Hospital; Prof John A. Eisman and Dr Nigel Morrison, Garvan Institute (1990-present).

Mechanism of UV radiation-induced immunosuppression: Prof. P. Hersey, Oncology and Immunology Unit, Royal Newcastle Hospital (1993-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, UV-visible detector, programmable fraction collector and chart recorder.

FUNDING in 1993 and 1994

NHMRC Direct and indirect effects Mason RS 1992

of estrogens in a human 1993 $44,644 bone formation model 1994 $45,155

NSWSCC Mechanisms of pigmentation Mason RS 1993 $44,726

and proliferation in response

to ultraviolet irradiation

Epidermal cell-derived cytokines Mason RS 1994 $33,341

and the adaptive responses to

UV irradiation

USCRF Hormone secretion in Mason RS 1993 $20,000

oncogenic osteomalacia

Total for 1993: $109,370

Total for 1994: $78,496

SCHOLARSHIPS in 1993 and 1994 PLEASE INSERT DETAILS

Name of scholarship awarded Name initials 1992-

etc ........

TEACHING in 1993

BMedSc: Human Life Sciences 2

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

Tutorials: 1, repeated 3 times, on endocrine physiology.

Tests: MCQs provided for informal assessment.

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

BMedSc: Human Life Sciences 3/Science 3

Lectures: 2 on lipid metabolism and development of atheroma

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

Examination: By essay-style question.

Dentistry 2/Science 2/Science Auxiliary

Lectures: 2, on mineral metabolism.

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 prac of 3 h, repeated 8 times, on endocrine tests, including glucose tolerance test, pregnancy test demonstration, and thyroid hormone radio- immunoassay, with a session on clinical features of hypo- and hyperthyroidism

Tutorials: 1.5 h follow-up session, repeated 4 times, involving respiratory physiology video with question and answer session following and general presentation and discussion of results of respiratory practical

Examination: by short answer questions.

Medicine 3 (Clinical Physiology - with Pharmacology)

Course Supervisor

Lectures: 4, on clinically-applied endocrine physiology.

Examination: by multiple choice and essay questions.

Medicine 4

Lecture: 1, on metabolic bone diseases.

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: by multiple choice questions (of the 5 alternatives, 1 correct variety).

Total distribution (Hours of formal teaching)

Dent2 Med2 Med3 Med4 Pha1 HLS2 HLS3/Sci3 Total

Lectures 2 18 4 1 10 4 4 43

Practical classes - 24 - - - - - 24

Tutorials - 12 - - 1 4 5 22

Total formal contact teaching time = 89 h

In addition time was spent on lecture preparation, student consultations, exam setting, exam marking, essay marking, Hons thesis marking, course supervisor duties and curriculum development.

OTHER ACTIVITIES in 1993

Refereeing

Manuscripts: for Clinical Chemistry (1), Journal of Bone and Mineral Research (1).

Grant applications: for NHMRC (3); Sandoz Foundation (2); MSD Research Foundation, Merck Sharp & Dohme (Aust) (1); Anti Cancer Foundation, South Australia (1).

PhD thesis examiner: for Univ. of Qld (1)

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 (1993-4)

University committees

Member, Special Studies Program Committee.

Academic Board

Nominee of Academic Board for appointment committees.

Faculty of Medicine committees

Member, Staff-student liason committee.

Vertical Stream Co-ordinator, Endocrinology and Metabolism.

Member, Year 3 Curriculum committee.

Member, Curriculum Working Party for 4 year degree course; Chair, subcommittee on integraton of basic and clinical sciences.

Member, Research Grants Standing Subcommittee of Postgraduate Committee in Medicine.

Community and hospital

Member, Working Party for development of a service plan for Endocrinology, Central Sydney Area Health Service (Bone and Mineral Working Group).

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

5-YEAR RESEARCH PUBLICATIONS

JOURNAL ARTICLES

1989

Cheng CL, Ma J, Wu PC, Mason RS, Posen S (1989) Osteomalacia secondary to osteosarcoma: a case report. Journal of Bone Joint Surgery, 71A, 288-292

Diamond T, Stiel D, Mason RS, Lissner D, Bikle D, Wilson S, Posen S (1989) Serum vitamin D metabolites are not responsible for low turnover osteoporosis in chronic liver disease. Journal of Clinical Endocrinology Metabolism, 69, 1234-1239

1990

Pryke AM, Duggan C, White CP, Posen S, Mason RS (1990) Tumor necrosis factor-alpha induces vitamin D 1-hydroxylase activity in normal human alveolar macrophages. Journal of Cell Physiology, 142:652-656

1991

Slater M (1991) A method for the examination of the same cell using light, scanning and transmission electron microscopy. Biotechnic and Histochemistry (formerly Stain Technology), 1, 63-69

Slater M (1991) Differential silver enhanced double labelling in immuno electronmicroscopy. Biotechnic and Histochemistry, 1, 153-154

Slater M (1991) Plurihormonality in the secretory granules of the normal human pituitary: An immuno electronmicroscopic study. Experientia, 47, 267-270

1992

Wong SYP, Slater SR, Evans RA, Mason RS, Lancaster EK, Acland SM, Eade Y, Hills E, Dunstan CR (1992) Metabolic studies in kidney stone disease. Quarterly Journal of Medicine, 82, 247-258

Haughton MA, Mason RS (1992) Immunonephelometric assay of vitamin D binding protein. Clinical Chemistry, 38, 1796-1801

1993

Dissanayake N, Greenoak G, Mason RS (1993) Effects of UV irradiation on human skin-derived epidermal cells in vitro. Journal of Cellular Physiology, 157, 119-127

Slater M (1993) Multiple labeling with silver enhanced gold probes. Journal of Histotechnology, 16, 229-234

Early 1994

Slater M, Patava J, Mason RS (1994) The role of chondroitin sulphate glycosaminoglycans in mineralising osteoblast-like cells: Effects of hormonal manipulation. Journal of Bone and Mineral Research, 9, 161-169

Slater M, Mason RS (1994) Determination of comparable labeling densities in quantitative immunoelectron microscopic double labelling studies. Biotechnic & Histochemistry, 69, 1-9

McLeod SD, Ranson M, Mason RS (1994) Effects of estrogens on human melanocytes in vitro. Journal of Steroid Biochemistry and Molecular Biology, 49 (in press)

Slater M (1994) Ultrastructural multiple labeling using colloidal gold. Micron, 24, 661-675

INVITED REVIEWS IN UNREFEREED JOURNALS

Mason RS, Dissanayake N, McLeod SD (1991) Effects of sunlight on skin. Wisenet Journal, 26, 3-4

Kelly PJ, Mason RS, Eisman JA (1991) The investigation of calcium and bone disorders. Fellowship Affairs, Royal Australasian College of Physicians, 10, 23-26

CONFERENCE ABSTRACTS AND PRESENTATIONS in 1993

Patava J, Slater M, Kingham KM, Mason RS (1993) Characterisation of a novel model for the study of bone growth and its response to 17beta-estradiol. Proceedings of the Australian Society for Medical Research, NSW Branch Scientific Meeting, 15, Sydney (Jun)

McLeod SD, Smith C, Mason RS (1993) Stimulation of human tyrosinase by POMC-derived peptides. Proceedings of the Australian Society for Medical Research, NSW Branch Scientific Meeting, 45, Sydney (Jun) (Young Investigator Prize awarded to first-named, presenting author)

Slater M, Patava J, Mason RS (1993) Chondroitin sulphate glycosaminoglycans and mineral deposition in a human osteoblast-like model culture. Proceedings of the Australian Society for Medical Research, NSW Branch Scientific Meeting, 53, Sydney (Jun)

Slater M, Patava J, Mason RS (1993) Thrombospondin - a bone growth factor? Proceedings of the Australian and New Zealand Bone and Mineral Society, 26, Queenstown, NZ (Sep)

Mason RS, Slater M, Patava J (1993) Growth factors and bone. Proceedings of the Australian Society for Medical Research, Annual Scientific Meeting, abstract K4, Adelaide (Nov)

Dissanayake N, Mason RS (1993) Mechanisms of pigmentation and proliferation after UV irradiation. Proceedings of the NSW Cancer Council, Annual Research Seminar, 26, Sydney (Month?)