Calmodulin Is Essential for Estrogen Receptor Interaction with Its Motif and Activation of Responsive Promoter

Abstract

Calmodulin (CaM) has been reported to have affinity for the estrogen receptor (ER). Observations reported here reveal a direct physical interaction between purified CaM and ER. This direct ER-CaM interaction may be an initial event preceding the assembly of ER plus auxiliary proteins into the active ER complex with its DNA motif, the estrogen response element. We demonstrate that CaM is an integral component of this complex by using a system reconstituted from purified ER and nuclear extract from ER-negative breast cancer cells and also with ER-depleted nuclear extract of an ER-positive breast cancer cell line. Although CaM is essential for formation of this complex, it is not sufficient, suggesting roles also of auxiliary proteins. CaM also is functionally required for activation of an ER-responsive promoter, in the 17␤-estradiol-ER pathway of hormone action and regulation of 17␤-estradiol-responsive gene expression that is associated with proliferation of mammary epithelial cells. CaM1 plays a pivotal role in the proliferation of a variety of cells. Several observations indicate CaM involvement in estrogen regulation of breast cancer cell growth. Calcium homeostasis is lost (1), and expression of calcium binding proteins is modulated (2-4). There is an overall increase of Ca 2ϩ levels in human mammary tumors (5, 6), and CaM concentrations are 2-3-fold higher in estrogen receptor-positive (ERϩ) than in estrogen receptor-negative (ERϪ) breast tumors (7). The growth of breast cancer cells is highly sensitive to anti-calmodulin drugs (8, 9), and the anti-estrogen Tamoxifen binds to CaM with high affinity and antagonizes its action (10). CaM also modulates estrogen (17␤-estradiol (E2)) binding to ER (11), and synergistic inhibition of growth by CaM inhibitors and antiestrogens (12, 13) are associated with breast cancer.Both anti-estrogens and anti-calmodulin drugs block breast cancer cells at an identical point in the G 1 phase of the cell cycle (14). Furthermore, CaM is known to stimulate the phosphorylation of estrogen receptors (15). Biochemical evidence suggests an interaction of CaM with ER (11, 16 -19), but these results did not demonstrate a direct physical contact.We examined more directly the possibility of a role of CaM in key downstream events of E2 action, i.e. the interaction of ER protein with its cognate DNA sequence (estrogen response element (ERE)) to form the ER⅐ERE complex and the resulting transactivation of an E2-responsive promoter. We observed that CaM directly interacts with ER, and is not only an integral component of the ER⅐ERE complex but also plays an essential role in complex formation. Its functional involvement in the molecular pathway of E2-induced transactivation of hormoneresponsive genes in breast cancers was tested. EXPERIMENTAL PROCEDURESCell Lines and Culture-MCF-7 and MDA-MB-231 cell lines were obtained from ATCC and were grown in rich medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2.8 M hydrocortisone, 1 g/ml insulin, and 12.5 ng/ml epide...

Calmodulin-independent, agonistic properties of a peptide containing the calmodulin binding site of estrogen receptor α

“…the initial step of the transcription cycle. In contrast to CaM inhibitors which have been reported to impede ER-mediated transactivation (Biswas et al, 1998;Garcia Pedrero et al, 2002;Li et al, 2005), synthetic peptides containing the P295 -T311 sequence would confer to ER an activated conformation similar to that induced by CaM, even if they are unable to directly interact with CaM.…”

“…This elimination process facilitates the access of newly govern transcription and turnover rate of ER are closely interrelated Metivier et al, 2003;Yan et al, 2003;Laios et al, 2005). Unexpectedly CaM, while being an important determinant of ER-mediated transcription (Biswas et al, 1998;Garcia Pedrero et al, 2002;Li et al, 2003;Li et al, 2005) has been reported to protect ER against proteasomal degradation (Castoria et al, 1988;Li et al, 2001;Li et al, 2006). This paradox led us to explore further the mechanism of action of this co-regulator.…”

“…Calmodulin (CaM), an ubiquitous Ca ++ sensor protein, is one of these co-regulators, the importance of which in ER-mediated transcription is now well recognized (Biswas et al, 1998;Garcia Pedrero et al, 2002;Li et al, 2003;Li et al, 2005).…”

“…Early studies revealed that the association of CaM with ER enhances the binding of the latter to estrogen response elements (ERE) (Biswas et al, 1998;Bouhoute and Leclercq, 1995), a step of prime importance for initiating the transcription of estrogenregulated genes. There is indeed a general consensus that transcription occurs according to a well defined cyclic process involving the combinatorial and sequential recruitment of enzymes and factors required for the assembly of transcriptionally productive complexes Metivier et al, 2003;Shang et al, 2000).…”

Calmodulin, a regulatory partner of the estrogen receptor alpha in breast cancer cells

“…Early studies from our laboratory revealed that CaM increases the ability of the receptor to associate with estrogen response elements (EREs) (Bouhoute, Leclercq, 1995). Subsequent independent investigations (Biswas et al, 1998) indicated that it operates as a co-regulator of major importance for the formation of stable ERα~ERE complexes. The additional observations of these authors that a CaM antagonist (W7) abrogates the transactivation of an E 2 -responsive promoter led them to conclude that CaM participates to ERα-mediated transcription.…”