Estralita Martin, Ph.D., Research Interest

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Estralita Martin, Ph.D.

RESEARCH INTEREST

Six hormones - 1,25 vitamin D, parathyroid hormone (PTH), calcitonin (CT), calcitonin-gene-related peptide (CGRP), parathyroid hormone-related peptide (PTHrP) and amylin - are concerned with the regulation of calcium metabolism. The last three hormones regulate calcium metabolism to a lesser extent than the first three hormones. The primary action of 1,25 vitamin D is to increase calcium absorption from the intestine. PTH and PTHrP mobilize calcium from bone whereas CGRP and amylin share effects on calcium regulation with CT by inhibiting bone resorption. Tumor tissues have been shown to produce these as well as other hormones. Studies using these cells have indicated that some hormones alter the subsequent course of the malignant process, some alter the course of other tissues, and some may be used as markers for the illness. In fact, from this research, an important area of biology has arisen which concerns itself with understanding the implication of hormone synthesis and secretion by cancer cells.

This research has also led to dramatic alteration in our thinking with regard to the endocrine activity of tumors - especially the underlying events that lead to peptide hormone production by tumor cells - and to the interaction of such hormones with normal and neoplastic cell receptors. I have therefore undertaken studies which address the secretory process of the calcemic regulatory hormones by cancer cells. I have addressed the secretory process of these hormones by using cancer cells from two broad categories. The first set of cells, medullary thyroid carcinoma cells, represent a minority of tumors in terms of incidence and arise directly from tissues composed of well-recognized endocrine cells. The second set of cells encompasses most of the common parent cell having endocrine differentiation features (e.g., lung cancers of various cell types, especially small cell carcinoma). From these studies I have been able to demonstrate that although serum calcium levels are likely to be the primary physiologic regulator of the calcium regulatory hormones, these hormones are potential regulators of one another's secretion. I have demonstrated that (1) CT, CGRP, amylin and PTHrP regulate the secretion of CT, CGRP, and PTHrP from both pulmonary carcinoid cells and small cell lung cancer cells albeit differently and (2) amylin and PTHrP regulate the secretion of PTHrP and CGRP secretion from medullary thyroid carcinoma cells. This work has resulted in the submission of three papers (see curriculum vitae). Other publications are in preparation.

These same calcium regulatory hormones have been shown to be co-localized and co-secreted with chromogranin A (CgA), a 49, kilodalton protein postulated to have a role in the biosynthesis, storage, and secretion of its co-resident hormones. I have been able to demonstrate that peptides derived from CgA not only affect the regulated secretion of CgA, CT, and CGRP but also have specific secretory effects on proteins that are derived from the same precursor molecule in both types of cancer cells. Eight publications have resulted from this work (see curriculum vitae). More detailed publications have been either submitted or are in preparation. I am presently studying the effects of the CgA-derived peptides on the secretion of the remaining calcium regulatory hormones. I have also been able to demonstrate that at least three of the calcium regulatory hormones - amylin, CGRP, and CT - regulate the secretion of CgA in both cancer cell types. These results will be presented with the papers submitted regarding the effect of the calcemic regulatory hormones on one another's secretion.

The widespread occurrence of CgA, suggest an important biological role for this protein. Proposals for the functions of CgA include a role (1) in the packaging and sorting of regulatory peptides to the regulated pathway of secretion, (2) in the organization of the content of secretory granules, (3) as a carrier protein for regulatory peptides after secretion, (4) as precursors of regulatory peptides, and (5) as a protein which has some biological activity on target cells. No direct evidence in support of any of these hypotheses has been obtained so far. However, the most compelling hypothesis to date regarding the biological function of CgA is that it is processed by its resident endocrine glands to biologically active peptides that regulate the function of those glands.

Therefore, the importance of my studies lies in the fact that perhaps the best-defined qualitative changes occurring in hormones of endocrine tumors involve disorders of packaging of the peptide. The proper secretion of a bioactive, small peptide hormone clearly requires a complex series of events involving not only regulation of the expression of the genes for the hormones, but also the packaging steps in secretory granules that convert precursor forms to bioactive mature forms. This may be the role of CgA-derived peptides. These packaging processes, even in differentiated endocrine tumors, often fail to develop fully in neoplastic cells, as compared to the mature parent cells. This failure to achieve the fully mature endocrine phenotype probably explains the well-known phenomenon of finding increased quantities of large-molecular-weight precursor forms of peptide hormones in defined endocrine cancers.

My studies can also be used to determine quantitatively if in solid tumors, such as small cell carcinoma of the lung which has a relatively high proportion of cells with endocrine features, the predominant type of hormone secreted can also change with time. This change can even be manifested as a changing set of clinical syndromes in a given patient owing to the presence of different hormone excesses. For example patients with small cell lung carcinoma may present with the clinical features of excess vasopressin secretion, but later may develop the syndrome of excess ACTH production.

A second area of interest is the specific function of normal osteoblasts and osteoclasts. Bone is produced, maintained, remodeled, and modulated by a complex array of local cell types, including osteocytes, osteoblasts, osteoclasts, and mesenchymal elements. The biochemical and molecular mechanisms that control bone remodeling have been explored in vivo and in vitro in whole bone, organ, primary cell, and transformed cell culture systems using conventional histological, biochemical and molecular biological approaches. All these studies of bone and bone cells, however, have been complicated by the complexity and cellular heterogeneity of the tissue. This inherent problem has resulted in the continuing accumulation of contradictory data about even the most fundamental properties of bone cells. Thus, an elucidation of the molecular events involved in hormonal regulation of bone metabolism would be aided by the identification of individual cells that secrete specific osteoblastic products and by direct quantification of these products. My goal is to investigate new and defined functions of osteoblasts within a heterogeneous population of bone derived cells by using two novel methods, the reverse hemolytic plaque assay (RHPA) and the cell-blotting assay (CBA) in conjunction with in situ hybridization. I plan to accomplish this by correlating and monitoring the production and secretion of the classical osteoblast products (alkaline phosphatase, BGP, and collagen type I) with the newly-define osteoblast products (GMF-CSF, PDGF, IGF-1, TGF beta, and IL-1) from cells isolated from the long bone of rats following hormonal treatment (PTH, CT, 1,25 dihydroxyvitamin D, estrogen, and testosterone). These studies would be conducted on neonatal rat. Preliminary data has resulted in one publication (see curriculum vitae). It is important to note, however, that the RHPA could be applied to studies on human osteoclasts.

The effect of each of the hormones I plan to study, except for testosterone, on osteoblast metabolism has been extensively investigated (for review see Skjodt and Russell, 1992 in Gowen M [ed] Cytokines and Bone Metabolism. CRC Press, Inc. Boca Raton, FL pp. 1-70). PTH has been shown to increase and decrease both alkaline phosphatase and collagen production as well as to decrease 1,25 dihydroxyvitamin D stimulated BGP transcription. 1,25 dihydroxyvitamin D has been shown to increase alkaline phosphatase, collagen type I, BGP, matrix gla protein, osteonectin, and osteopontin synthesis. CT increases alkaline phosphatase and collagen type I synthesis and IGF-1 production. Estrogen has been shown to up and down regulate alkaline phosphatase, BGP, IGF-1, and osteonectin synthesis, as well as procollagen type I and TGF transcription. I feel that in order to have a better understanding of the various hormonal effects on osteoblast metabolism, one needs to look not at only the one or two osteoblast products that have been shown to be affected but the other products as well since they appear to be interconnected. Also, since the secretion of one hormone usually affects the secretion of another, it is important to investigate how one hormone affects the production/secretion of osteoblast products caused by another hormone. Therefore, it would be advantageous to do studies that incorporate combinations of hormones (resulting in a more physiological investigation) and allow one to study multiple osteoblast products at the same time with the same cell. I realize this appears at face value to be a sizable undertaking. Once again the advantages of RHPA and CBA methodologies along with in situ hybridization come into play. First, few cells are needed to do each experiment. Secondly, studies regarding the functions of single cells within a mixed cell population in a sensitive and specific manner can be undertaken. Thirdly, I will be able to study the affects of combinations of hormones on multiple osteoblast products. Fourthly, I can directly quantitate cellular functions.

These studies can then be tied into my third area of interest - a collaborative study on the management of musculoskeletal complications of spinal cord injury. The purpose of this research is to develop clinical assessment procedures and to provide a panel of biochemical and immunochemical tests that can be applied along with other modalities, such as bone densitometry, in the diagnosis and management of the musculoskeletal complications of patients with spinal cord disease and injury. The bone markers we have developed and use are immunochemically based. They are classical and novel immunoassays for the respective bone proteins under study. Thus, in addition to standard immunoassays, they also include new immunoassay formats that allow the precise identification in serum of the bone proteins under study, such as bone alkaline phosphatase (BAP), new skeletal markers, such as Gla protein (BGP, osteocalcin) and its derived peptides, and parathyroid hormone (PTH) and calcitonin (CT). We have applied these immunoassays along with bone densitometry studies to access skeletal changes in our patients. These data will be used to prevent osteoporosis/fratures following spinal cord injury.

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