Regulation of the Rhox5 Homeobox Gene in Primary Granulosa Cells: Preovulatory Expression and Dependence on SP1/SP3 and GABP1

Abstract

Homeobox genes encode transcription factors that regulate embryonic development and postnatal events. Rhox5 (previously called Pem), the founding member of a homeobox gene cluster that we recently identified on the X chromosome, is selectively expressed in granulosa cells in the ovary and other somatic-cell types in other reproductive organs. In this report, we investigate its regulation in granulosa cells in the rat ovary. We found that Rhox5 expression in the ovary is governed by the Rhox5 distal promoter and is expressed at least as early as Day 5 postpartum. Rhox5 mRNA levels are regulated during the ovarian cycle, peaking before ovulation. Deletion analysis revealed a 25-nt element essential for distal promoter transcription in primary granulosa cells. This distal promoter element contains two ETS and one SP1 transcription-factor family binding sites that mutagenesis analysis indicated were essential for high-level transcription. This element was both necessary and sufficient for transcription, because it activated transcription when placed upstream of a heterologous minimal promoter. Cold competition and electrophoretic mobility shift assay studies demonstrated that SP1, SP3, and the ETS family transcription factor GABP bound this element. Dominant-negative forms of GABP and SP3 repressed distal promoter expression in primary rat granulosa, showing that these factors are crucial for Rhox5 expression. Cotransfection of dominant-negative mutants indicated that Rhox5 expression in granulosa cells is regulated by the c-Jun N-terminal protein kinase (JNK, MAPK8) and RAS pathways, which are known to be upstream of ETS family transcription factors. The discovery that Rhox5 expression in granulosa cells is regulated by MAPK pathways and ETS and SP1 family members provides an opportunity to understand how these regulatory pathways and factors collaborate to regulate gene expression during the ovarian cycle.

The Rhox genes

“…K-RAS signaling is also required for the phosphorylation and activation of MAPK3/1 (ERK1/2), which, in turn, is required for granulosa cells to promote LH-induced resumption of germ cell meiosis, ovulation, and luteinization (Fan et al 2009). Gonadotropin treatment induces peak MAPK3/1 activity in rodent granulosa cells at a similar time point as that for maximal Pd mRNA levels (MacLean et al 2005b, Fan et al 2009), suggesting the possibility that Pd transcription also requires MAPK3/1. While this has not been tested, it is known that other components of the MAP kinase pathway -namely the stress-activated protein kinase/c-Jun amino-terminal kinases -are required for Pd transcription (Rao et al 2002a, MacLean et al 2005b).…”

“…Subsequent work demonstrated that Rhox5 is not only expressed in a subset of T-cell lymphomas, but it is widely expressed in tumors derived from many different cell types and tissue origins (Wilkinson et al 1990, Maiti et al 1996b, Wayne et al 2002a. Its ubiquitous expression in tumors may derive, in part, from its ability to be induced by the Ras proto-oncogene (Rao et al 2002a, MacLean et al 2005b. RHOX5 may also have a causal role in tumor formation, as it induces an immune response in mouse tumors (Ono et al 2000), and it physically interacts with proteins involved in malignancy (Lemmens et al 2001, Guo et al 2005).…”

“…The Pd is expressed in the ovary and placenta, and is also co-expressed with the Pp in the testes (Maiti et al 1996a, 2001, MacLean et al 2005b. The likely cellular site of Pd expression in the ovary is mural granulosa cells, as that is where RHOX5 protein has been shown to be specifically expressed (Pitman et al 1998).…”

“…A minimal element required for Pd transcription has been defined (w100 nt upstream of its transcription start site) that is only 22 nt in length (Rao et al 2002a, MacLean et al 2005b). This 22-nt element is sufficient to activate the transcription of an inactive heterologous promoter in primary granulosa cells and tumor cells.…”

“…A key signal transduction event required for Pd transcripion is initiated by the G-protein RAS (Rao et al 2002a, MacLean et al 2005b). Gain-of-function evidence for this is that transient transfection of a constitutive form of RAS induces Pd expression in primary granulosa cells, mesenchymal stem cells, and tumor cells.…”