Elsevier

Bone

Volume 34, Issue 1, January 2004, Pages 100-111
Bone

Interaction of estrogen receptor α with protein kinase C α and c-Src in osteoblasts during differentiation

https://doi.org/10.1016/j.bone.2003.09.007Get rights and content

Abstract

In cultured osteoblasts, protein kinase C (PKC) activity increases and estrogen receptor α (ERα) binding capacity decreases upon confluence. We investigated potential interactions between ERα and PKC isoforms and their confluence-induced modulations in clonal ROS.SMER#14 cells and primary osteoblasts. In sub-confluent ROS.SMER#14 cells, which express an exogenous plus small amounts of the endogenous ERα gene, the receptor appeared as two main bands of ≈66 and ≈46 kDa. In over-confluent, more differentiated cells, the cytosolic ≈66 kDa ERα appeared decreased and the ≈46 kDa variant increased. Enhanced expression and/or membrane translocation of PKCα and PKCε, but not PKCζ, was evidenced at over-confluence, along with transient increases in expression and kinase activity of c-Src, accompanied by membrane translocation of the kinase-activated enzyme. In contrast, negligible membrane translocation of PKCα and/or activated c-Src was observed in parental ROS 17/2.8 cells, which express low levels of full-length ERα. PKCα from over-confluent cells phosphorylated p60c-Src in vitro, suggesting functional interaction between the two kinases. ERα co-immunoprecipitated c-Src and PKCα, mostly in its cleaved form (PKMα). An analogous interaction was observed in primary osteoblasts. However, in these cells, much more PKCα/PKMα was ERα-co-immunoprecipitated at over-confluence, a condition in which the shorter, ≈46 kDa ERα variant is increased. This interaction was enhanced by estradiol treatment or PKC down-regulation, but was unaffected by c-Src inhibition. These data highlight direct PKCα–c-Src–ERα interactions, which may be crucial in the modulation of estrogen responsiveness and the differentiation process in osteoblasts.

Introduction

The relevance of estrogens in bone homeostasis is demonstrated by the significant loss in bone mass after menopause, eventually leading to osteoporosis [1]. Increasing evidence indicates that reduced estrogen receptor α (ERα) levels and/or activity represent a cause of the weaker response to estrogens in many cell types, including osteoblasts [2], [3]. However, the underlying molecular mechanisms are not yet fully understood [4].

Protein kinase C (PKC) is a serine/threonine kinase family involved in a variety of cell functions, including the processes of proliferation and differentiation [5], [6], [7], [8]. PKC isoenzymes differ remarkably in their structure, tissue distribution, mode of activation, and substrate specificity [9], and are classified in three groups: conventional, or calcium- and diacylglycerol-dependent PKCs (α, βI, βII, and γ); novel, or calcium-independent but diacylglycerol-dependent PKCs (δ, ε, η, and θ); and atypical, or calcium- and diacylglycerol-independent PKCs (ζ and é/λ). The membrane spanning PKCμ is often placed in a separate class [10]. Specific roles in cell cycle progression and in differentiation have been hypothesized for different PKC isoforms [11]. For instance, PKCα and PKCζ appear to be involved in the differentiation of the K562 human erytroleukemia cells into megakaryocytes upon exposure to phorbol 12-myristil 13-acetate (PMA) [12]. PKCα and PKCδ are tightly involved in the differentiation of the 32D myeloid cell line [13]. In addition, changes in PKCα, PKC-ε, and PKC-ζ expression and activity are observed in human craniosynostoses [14], [15], [16], a disease characterized by accelerated osteoblast differentiation. PKCs also regulate ERα activity. Exogenous activation of PKC by phorbol esters down-regulates ERα in MCF-7 cells, and decreases estrogen responsiveness in osteoblast-like cells [3], [17], [18]. However, no direct molecular interaction has been described so far between PKCs and ERα.

Another kinase which may regulate ERα activity is c-Src, a nonreceptor tyrosine kinase [19] which has been described to phosphorylate ERα [19]. ERα alleles harboring missense mutations at Y537 [20], [21] give raise to constitutively active receptors. c-Src is also a substrate for PKC. Human c-Src is phosphorylated upon PMA exposure at serine-12 [22], a residue positioned in a conserved domain common to various PKC substrates. However, the physiologic significance of this phosphorylation is still unknown. To date, a direct association between Src and PKC has been demonstrated only for PKCδ in v-Src transformed cells [23]. This association occurs in the presence of an intact serine-12 phosphorylation site in Src, and results in intermolecular phosphorylations between the two kinases. PKCα also directly interacts with either Lyn or Src in the RBL-2H3 mast cell line, resulting in phosphorylation and increased activity of both tyrosine kinases [24].

Alterations of ERα, PKC, and c-Src pathways are involved in the pathogenesis of several bone diseases, including osteoporosis [25], [26], [27]. For instance, ERα-positive mammary tumors frequently metastasize to bone [28], [29], enhanced expression of PKCs is found in craniosynostoses [14], [15], and c-Src deletion induces osteopetrosis in mice [30], [31]. In all these diseases, osteoblast alterations have been observed. Therefore, we investigated the functional and molecular interactions among ERα, PKC, and c-Src with the aim of further highlighting their relevance in osteoblast biology [37].

Section snippets

Materials

Fetal bovine serum (FBS), glutamine, and penicillin/streptomycin were purchased from HyClone (Cramlington, NE). Polyclonal antibodies against PKC isoforms were either produced in our laboratory [22] or were purchased, along with the anti-ERα antibody (H184), all secondary antibodies, and protein G plus-agarose, from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal antibodies were used for PKCα (cat. #610107, BD Transduction Laboratories) and c-Src (cat. #OP07, Oncogene Res.

Alkaline phosphatase (ALP) activity assay

Triplicate aliquots of 0.1% SDS cell lysates were used for biochemical evaluation of ALP activity using the Sigma-Aldrich kit no. 104, according to the manufacturer's instruction.

ROS.SMER#14 cells undergo spontaneous differentiation at confluence

ROS.SMER#14 cells are rat osteosarcoma-derived cells with an osteoblast phenotype, in which the ERα expression is returned to physiologic levels by stable transfection. These cells express approximately 2500–3000 estrogen binding sites per cell when sub-confluent and actively proliferating. At over-confluence, proliferation is nearly abolished, and the estrogen binding sites decrease to approximately 200 per cell despite unchanged ERα mRNA levels. As a consequence, at this stage, estrogen

Discussion

In this study, functional interactions among PKCα, c-Src, and ERα have been shown to occur in osteoblast-like cells and primary osteoblasts, along with modulations of ERα protein variants at over-confluence, a condition whereby exit of osteoblasts from cell cycle and the acquisition of a more differentiated phenotype are achieved [3], [33].

Estrogen sensitivity in osteoblasts represents a critical requirement for physiologic bone homeostasis. The ERα is highly expressed in these cells, where it

Acknowledgements

We warmly thank Prof. Goeffrey Greene, Chicago University, who kindly donated the H222 antibody. This study was funded by the Agenzia Spaziale Italiana (ASI) to S.M, and by the Consiglio Nazionale delle Ricerche (CNR), Ministero Università e Ricerca (MIUR) and European Union project GENOSPORA, contract no. QLK6-1999-02108 to A.T. This study was also partially supported by the Telethon Grant #831 and by grants from Associazione Italiana Ricerca Cancro (AIRC), and Agenzia Spaziale Italiana (ASI)

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