Analysis of gene expression in PTHrP^−/− mammary buds supports a role for BMP signaling and MMP2 in the initiation of ductal morphogenesis

Abstract

Parathyroid hormone-related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP 2/2 mammary buds, and in WT and K14-PTHrP ventral skin at E15.5. By cross-referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds.

Molecular Mechanisms Guiding Embryonic Mammary Gland Development

“…There is also a loss of sexual dimorphism because PTHrP from the epithelial cells is necessary for the expression of androgen receptor (AR) in the mesenchyme. In addition to the AR, PTHrP signaling has now been shown to regulate the expression of at least 40 different molecules in the mammary mesenchyme, although many of these may likely require coexpression of ventrally restricted BMP4 to be induced by PTHrP (Dunbar et al 1999;Foley et al 2001;Hens et al 2007;Hens et al 2009). These studies underscore the significance of epithelial/mesenchymal crosstalk in the formation of the mammary bud and show the importance of PTHrP as an epithelial signal that shapes the nature of the primary mesenchyme.…”

“…These data suggest that autocrine BMP4 acts downstream of PTHrP in the mammary mesenchyme to trigger the initiation of ductal morphogenesis. More recently, analysis of gene expression changes in PTHrP 2/2 versus WT mammary buds at E15 identified 35 genes that were regulated by PTHrP signaling in the primary mammary mesenchyme (Hens et al 2009). Of these 35 genes, at least six are known targets of BMP signaling, reinforcing the notion that PTHrP action in these cells leads to activation of the BMP pathway.…”

“…Mesenchymal expression of Msx2 contributes to the lateral inhibition of hair follicle formation around the primary duct and nipple and it may contribute to ductal outgrowth as well. The combination of BMP4 and PTHrP also activates matrix metalloproteinase 2 (MMP2) activity in mesenchymal cells and MMP inhibitors have been shown to block PTHrPdependent mammary bud outgrowth in culture (Hens et al 2009). However, it should be noted that ductal outgrowth occurs in MMP2 2/2 mice, so that although MMP2 activity likely participates in ductal outgrowth in vivo, it is not required (Wiseman et al 2003).…”

Parathyroid hormone-related protein activates Wnt signaling to specify the embryonic mammary mesenchyme

“…RT-PCR reactions were performed by assaying each pool in triplicate using the Full Velocity or Brilliant SYBR Green qPCR Kit (Agilent Technologies, Santa Clara, CA, USA) using the following primers: Rspo1F, 5Ј-CGACATGAAC -AAATGCATCA-3Ј; Rspo1R, 5Ј-CTCCTGACACTTGGTGCAGA-3Ј; WNT11F, 5Ј-CCACCATCAGTCACACCATC-3Ј; and WNT11R, 5Ј-TTTATTGGCTTGGGATCCTG-3Ј. Samples were normalized for relative quantification of expression by the 2 - CT method using Gapdh as an internal control (Hens et al, 2009). For each assay, gene expression in the WT samples was arbitrarily set to 1.…”

“…In addition, a prior gene array study suggested that PTHrP might regulate the expression of Rspo1 in the mammary mesenchyme (Hens et al, 2009). Rspo1 is a member of the R-spondin superfamily of proteins that have been shown to enhance Wnt signaling at sites of native Wnt expression.…”