A Comparative Study of Estrogen Receptor Signaling under the Influence of CDRI-99/373 and SERMs

Abstract

Estrogens are critical regulators of reproductive physiology and are also associated with non-reproductive effects, some of which are beneficial like improved cognitive performance and bone maintenance whereas some are detrimental which include increased incidences of breast cancer, endometriosis, endometrial cancer, venous thrombemboli and stroke (Grady et al., 1995). The biological actions of estrogens are mediated by estrogen binding to one of two specific estrogen receptors (ERs), ERα and ERβ, the ligand-regulated transcription factors. ERα and ERβ have different transcriptional activities in certain ligand, cell-type, and promoter contexts (Rachez and Freedman, 2001). ERs regulate gene expression by binding to their cognate response element or through protein–protein interactions with other transcription factors (Paech et al., 1997). ERα and ERβ share the highest degree of sequence identity within their DNA-binding domains (DBDs) which is followed by the ligand binding domain (LBD). Estrogen deficiency due to menopause or other reason is implicated in a number of pathological conditions, including osteoporosis. It is now recognized that E2 prevents bone loss via multiple and complex effects on bone marrow and bone cells, antiresorptive action (Riggs et al., 1998), resulting in decreased osteoclast (OC) formation, increased OC apoptosis, and decreased capacity of mature OCs to resorb bone (Manolagas and Jilka, 1995). Osteoclast precursors seem to be direct targets of estrogen. E2 inhibits the expression levels of the cytokines, IL-1, tumor necrosis factor (TNF), and IL-6 (Roodman, 1999) that are involved in bone resorption (Ernst and Rodan, 1991). Effects of E2 on osteoclasts appear to be indirect, through elaboration of regulatory factors such as OPG by osteoblasts or by bone marrow stromal cells. Selective estrogen receptor modulators (SERMs) have come up as a rescue to the undesirable effects of estrogen replacement therapy for the management of post-menopausal osteoporosis. SERMs inhibit estrogen activation of the estrogen receptor (ER) in some tissues but activate ER in other. The tissue specific action of SERMs can be explained on the basis of the structure, the type of ER it binds to, and the set of molecules that interact with the ER-SERM complex in a particular cell (Dutertre and Smith, 2000). Raloxifene is the only SERM in clinical use for prevention of post-menopausal osteoporosis. It mimics the action of estrogen on the bone and liver but aantagonizes the effects of E2 in the breast and uterus (Black et al., 1994; Bryant, 2001). Unfortunately, long term use of raloxifene is known to cause incidence of hot flashes, deep vein thrombosis, pulmonary embolism and leg cramps, similar to that associated with the use of ERT (Fisher et al., 1998; Cummings et al., 1999). Thus, a need exists for effective therapeutic agent for prevention of osteoporosis with a more desirable benefit: risk profile. Recently, in a quest to develop tissue specific agents for estrogen related disorders without having inducible effects on uterus, CDRI’s efforts have led to the identification of novel antiresorptive agent viz. CDRI-99/373 (99/373). It prevents ovariectomy-induced bone loss in rats. A potent anti-osteoclastogenic activity has been described for its bone protective effects. However the molecular mechanisms underlying its bone preserving activity are not fully understood. The studies given in this dissertation were designed with a objective to evaluate the effect of 99/373 on estrogen signaling both in vitro and in vivo, in uterus, breast cancer cells and also during osteoclastogenic differentiation in murine macrophage cells with a view to compare with the available antiosteoporotic drug Raloxifene.