Transforming Growth Factor β Regulates Parathyroid Hormone-related Protein Expression in MDA-MB-231 Breast Cancer Cells through a Novel Smad/Ets Synergism

Abstract

The majority of breast cancers metastasizing to bone secrete parathyroid hormone-related protein (PTHrP). PTHrP induces local osteolysis that leads to activation of bone matrix-borne transforming growth factor ␤ (TGF␤). In turn, TGF␤ stimulates PTHrP expression and, thereby, accelerates bone destruction. We studied the mechanism by which TGF␤ activates PTHrP in invasive MDA-MB-231 breast cancer cells. We demonstrate that TGF␤1 up-regulates specifically the level of PTHrP P3 promoter-derived RNA in an actinomycin D-sensitive fashion. Transient transfection studies revealed that TGF␤1 and its effector Smad3 are able to activate the P3 promoter. This effect depended upon an AGAC box and a previously described Ets binding site. Addition of Ets1 greatly enhanced the Smad3/TGF␤-mediated activation. Ets2 had also some effect, whereas other Ets proteins, Elf-1, Ese-1, and Erf-1, failed to cooperate with Smad3. In comparison, Ets1 did not increase Smad3/TGF␤-induced stimulation of the TGF␤-responsive plasminogen activator inhibitor 1 (PAI-1) promoter. Smad3 and Smad4 were able to specifically interact with the PTHrP P3-AGAC box and to bind to the P3 promoter together with Ets1. Inhibition of endogenous Ets1 expression by calphostin C abrogated TGF␤-induced up-regulation of the P3 transcript, whereas it did not affect the TGF␤ effect on PAI expression. In TGF␤ receptor II-and Ets1-deficient, noninvasive MCF-7 breast cancer cells, TGF␤1 neither influenced endogenous PTHrP expression nor stimulated the PTHrP P3 promoter. These data suggest that TGF␤ activates PTHrP expression by specifically up-regulating transcription from the PTHrP P3 promoter through a novel Smad3/Ets1 synergism.Parathyroid hormone-related protein (PTHrP)

Ets2 and protein kinase Cepsilon are important regulators of parathyroid hormone-related protein expression in MCF-7 breast cancer cells

“…We have recently shown that TGFβ stimulates PTHrP expression in invasive MDA-MB-231 cells by inducing an Ets1/Smad3 co-operative effect on the PTHrP P3 promoter [20]. In the present study, we examined the mechanism of PTHrP regulation in non-invasive MCF-7 cells.…”

“…For PTHrP P3 promoter analysis by transient transfection assay, a − 328/+ 20 promoter fragment cloned upstream of a luciferase gene was used [20]. Etsbinding site, CCCACC element and AGAC box mutants of the P3 promoter were as described previously [18][19][20]. pcDNA3-based expression plasmids were used for the expression of Ets1, Ets2, Elf-1 and Ese-1 [20].…”

“…been identified [18][19][20], which contribute to the regulation of the promoter by transforming growth factor β (TGFβ), human Tcell lymphotrophic virus I-transactivator of the X region (HTLV-I-Tax), retinoic acid and adenovirus early region 1A protein [20][21][22][23].…”

“…Ets1 and Ets2 are targets of proto-oncogenic Ras that superactivate these proteins by inducing extracellular-signalregulated kinase (ERK) 1/2-dependent phosphorylation of a specific N-terminal threonine residue [33]. In contrast with Ets2, Ets1 is expressed only in invasive cancer cells [20,30,31].…”

TGFβ1 regulates HGF‐induced cell migration and hepatocyte growth factor receptor MET expression via C‐ets‐1 and miR‐128‐3p in basal‐like breast cancer

“…Several transcription factors were found to have predicted binding sites in the MET promoter (Table S4). C‐ets‐1 was the top candidate as this transcription factor has previously been described to synergize with SMAD3 (Lindemann et al ., 2001) and its expression also correlated the most with expression of TGFBR2 as well as of MET in breast cancer cell lines (Fig. 4A, B, Tables S3 and S5).…”