Prolactin-induced expression of vascular endothelial growth factor via Egr-1

Abstract

Angiogenesis is a dynamic process regulated by both local and systemic factors. Among these is vascular endothelial growth factor (VEGF), a potent effector of angiogenesis and vascular permeability. Previously we showed that VEGF is temporally and spatially regulated in the mouse mammary gland during development and lactation. Given the functions of prolactin (PRL) during these stages and the supporting role of the vasculature, we investigated the regulation of VEGF by PRL. Treatment of HC11 mouse mammary epithelial and Nb2 rat lymphoma cells with PRL induced VEGF expression. Deletion and mutation analysis identified a GC-rich region in the proximal region of the VEGF promoter that constitutively bound Sp1 and PRL-induced Egr-1. These sites conferred PRL-responsiveness leading to increased VEGF transcription. The induction of VEGF by PRL was PRL receptor-, Jak2- and MAP kinase kinase-dependent. Our results indicate that PRL induces VEGF expression through Egr-1, and implicates VEGF as an intermediary of PRL-regulated angiogenesis.

Introduction

Angiogenesis is a critical process that involves establishment of the microvasculature during normal organ development as well as during tumor initiation, progression and metastasis (Folkman, 2003). In particular, the mammary gland demonstrates extensive neovascularization and extravasation during postnatal development to ultimately support the nutritional and biochemical requirements of the secretory epithelium during lactation (Djonov et al., 2001). Likewise, it is well established that breast tumors require extensive neovascularization to facilitate their progression and survival (Weidner et al., 1991).

A major effector of mammary gland growth, function and tumorigenesis is prolactin (PRL). Derived from endocrine and autocrine/paracrine sources, PRL acts through different forms of the prolactin receptor (PRLR) to induce cell division and/or differentiation in normal or neoplastic epithelium (Vonderhaar, 1999). In addition to these effects, PRL has been implicated in the regulation of angiogenesis (Corbacho et al., 2002). Mature (23 kDa) PRL stimulates angiogenesis from differentiated, but not rapidly growing, vasculature in the chorioallantoic membrane assay (Struman et al., 1999). While some endothelial cells have been shown to express the PRLR (Merkle et al., 2000), others do not, raising the possibility that PRL regulates angiogenesis via a paracrine mechanism (Corbacho et al., 2002). In addition, several studies have demonstrated that proteolytic cleavage of PRL, potentially achieved through the actions of cathepsin D (Khurana et al., 1999, Clapp, 1987), gives rise to an N-terminal cleavage product (16 K PRL) that is antiangiogenic (Struman et al., 1999, Martini et al., 2000, D’Angelo et al., 1999, Clapp et al., 1993). Possibly acting through a separate form of the PRLR (Clapp et al., 1993), the 16 K fragment of PRL prevents vascular endothelial growth factor (VEGF)-induced translocation of Raf-1 to the plasma membrane and inhibits phosphorylation of Ras (D’Angelo et al., 1999), thereby antagonizing the activation of MAP kinase (D’Angelo et al., 1999).

Central to the establishment of a vascular supply are locally derived factors including VEGF that stimulate endothelial cells to proliferate (Houck et al., 1992) and become more permeable (Senger et al., 1986, Dvorak et al., 1999). Acting through its tyrosine kinase receptors, Flt-1 and Flk-1/KDR (Xie et al., 1999, Boocock et al., 1995), VEGF is frequently implicated as a paracrine and autocrine effector of angiogenesis (Ferrara and Davis-Smyth, 1997, De Jong et al., 1998). Multiple factors have been implicated in the regulation of VEGF expression in different tissues. Positive regulation occurs due to the effect of hypoxia on human mammary fibroblasts (Hlatky et al., 1994) and noradrenalin on rat brown adipocytes (Tonello et al., 1999). Retinoic acid induces VEGF via Sp1 and Sp3 in brionchiocarcinoma cells (Maeno et al., 2002), while epidermal growth factor (EGF) family members upregulate VEGF expression in human malignant glioma (Goldman et al., 1993) and epidermoid carcinoma cells (Gille et al., 1997). Similarly, VEGF is upregulated as a result of heregulin-induced over-expression of HER2/neu receptors in MCF-7 cells (Laughner et al., 2001). Furthermore, steroid hormones that are principal regulators of breast development and carcinogenesis also induce VEGF. Breast cancer cell lines (Stoner et al., 2004, Ruohola et al., 1999) and DMBA-induced mammary tumors (Nakamura et al., 1996) upregulate VEGF expression when exposed to estrogen. In addition, estrogen induces VEGF in cultured human endometrial cells (Shifren et al., 1996), in rat uterus (Cullinan-Bove and Koos, 1993) and in human endometrial adenocarcinoma cells (Mueller et al., 2000). More recently it was also demonstrated that progesterone induces VEGF expression in T47D breast cancer cells (Wu et al., 2004). Regulation of VEGF gene transcription occurs as the result of several factors acting on the VEGF promoter including classical estrogen response elements (Mueller et al., 2000), Sp1 and Sp3 (Finkenzeller et al., 1997), AP2 (Gille et al., 1997) and STAT3 (Niu et al., 2002).

Recently we detailed the spatio-temporal distribution of VEGF mRNA and protein, and its receptors, within the developing mouse mammary gland, and identified that VEGF₁₆₄ and VEGF₁₂₀ gene expression increased in the mammary epithelium at the onset of lactation (Hovey et al., 2001). Given this observation and evidence implicating PRL in the regulation of angiogenesis, we have examined the role for PRL in the regulation of VEGF transcription. We report that PRL regulates VEGF gene transcription via a PRLR, Jak2 and MAP kinase kinase-dependent mechanism and elicits its effect through the action of Egr-1 on the VEGF promoter.