John J. Wysolmerski, M.D.

Featured Investigator and Recipient of a Pilot and Feasibility Project Award

John J. WysolmerskiJohn J. Wysolmerski 

John J. Wysolmerski, M.D.is an Associate Professor of Medicine.

Research Summary

My laboratory is interested in the functions of parathyroid hormone-related protein (PTHrP) during normal mammary gland development and physiology. Soon after PTHrP was discovered, it was found to be expressed in the breast and secreted into milk in large quantities. In fact, breast milk is the most abundant source of PTHrP in nature. We now realize that PTHrP has a remarkable relationship with the breast, apparently having different functions at all the various stages of the mammary life cycle. My group has focused its efforts on elucidating the functions of PTHrP during mammary development in embryos and on the function(s) of PTHrP during lactation.

We had previously shown that PTHrP and the Type I PTH/PTHrP receptor (PTHR1) are both necessary for the formation of mammary glands and nipples in mouse and human embryos. PTHrP is expressed in mammary epithelial cells soon after the mammary bud forms. The PTHR1 is expressed in the mesenchymal cells underneath the embryonic epidermis and surrounding the developing mammary bud. Experiments from our laboratory have demonstrated that PTHrP signaling triggers a series of cell fate choices necessary for the formation of mammary structures. PTHrP induces the mesenchymal cells near the epithelial bud to become mammary specific. These cells, in turn, maintain the mammary (as opposed to epidermal) fate of the epithelial cells and lead to the outgrowth of the epithelial bud, the initiation of ductal branching morphogenesis and the differentiation of the nearby skin into the nipple sheath. We are currently investigating the mechanisms by which PTHrP signaling leads to the differentiation of the mammary mesenchyme. We have found that PTHrP induces the expression of LEF1, b-catenin and a specific cadherin in the mesenchymal cells around the mammary bud. These data suggest that PTHrP signaling may interact in some way with the canonical wnt-signaling pathway. We are investigating this possibility by examining the effects of PTHrP signaling on wnt signaling both in vivo and in vitro, For our studies in vivo, we are utilizing TOPGAL mice. These transgenic mice were engineered in the laboratory of Dr. Elaine Fuchs and contain TCF/b-catenin response elements driving the expression of the beta-galactosidase gene. We are currently using these mice to examine the pattern of canonical wnt-signaling during normal mammary development. We are also breeding this transgene into PTHrP and PTHR1 knockout mice in order to determine if loss of PTHrP signaling alters the normal pattern of wnt signaling or expression during embryonic mammary development. For our studies in vitro, we are examining the effects of PTHrP signaling on wnt-signaling and adipocyte differentiation in 3T3L1 cells. These experiments should allow us to determine if PTHrP signaling does interact with the wnt signaling pathway so that future experiments can address the mechanisms by which these pathways might interact to induce mammary mesenchyme differentiation.

Our second area of interest concerns the function of PTHrP during lactation. As noted above, PTHrP is expressed at high levels in mammary epithelial cells during lactation and it is secreted into milk in great quantities. There has been a great deal of speculation surrounding the possibility that PTHrP might participate in systemic calcium metabolism during lactation. Milk production places great stress on maternal calcium homeostasis and nursing mothers secrete 300 – 400 mg of calcium into milk per day. Much of this calcium comes from the maternal skeleton and lactation is a period of high bone turnover and rapid bone loss. Although these facts are well established, the mechanisms regulating this skeletal calcium efflux are poorly understood. Because it is made in the breast during lactation and because it has the ability to increase systemic bone resorption, several investigators have suggested that PTHrP might be secreted into the circulation and contribute to the increase in bone resorption during lactation. In order to test this idea, we have created a mouse model in which the PTHrP gene is specifically deleted only in the mammary gland and only at the onset of lactation. We made use of the bacterial Cre-lox recombinase system to delete the PTHrP gene. In the mammary gland one can take advantage of milk protein gene promoters to drive Cre expression. We chose to use the ovine beta-lactoglobulin promoter which is expressed only in mammary epithelial cells and only at the end of pregnancy and during lactation. We bred a BLG-Cre transgene onto a PTHrPlox/- background. The resulting mice lose their one copy of the PTHrP gene at the transition between pregnancy and lactation. Our analysis thus far has demonstrated that these mice fail to express PTHrP mRNA in their lactating mammary glands and their milk is devoid of PTHrP, indicating that we have been successful in deleting the PTHrP gene during lactation. We are currently examining the effects of deleting the PTHrP gene on maternal calcium metabolism. Data thus far demonstrate that the mammary gland structure is normal and that the mice suckle their young successfully, indicating that PTHrP is not necessary for lactation per se. However, our preliminary analysis suggests that the Cre-lox mice have lower levels of circulating PTHrP, lower rates of bone turnover, and higher bone mass. These data, if they are confirmed, would suggest that some PTHrP from the mammary gland does get secreted into the circulation and does participate in the mobilization of skeletal calcium stores in the lactating mothers. An additional finding from these studies is that pups drinking milk without PTHrP appear to have a higher total ash calcium content. Ongoing studies, using these mice will help to elucidate the exact role of PTHrP in maternal and neonatal calcium metabolism during lactation.

In the mammary gland one can take advantage of milk protein gene promoters to drive Cre expression. We chose to use the ovine beta-lactoglobulin promoter which is expressed only in mammary epithelial cells and only at the end of pregnancy and during lactation. We bred a BLG-Cre transgene onto a PTHrPlox/- background. The resulting mice lose their one copy of the PTHrP gene at the transition between pregnancy and lactation. Our analysis thus far has demonstrated that these mice fail to express PTHrP mRNA in their lactating mammary glands and their milk is devoid of PTHrP, indicating that we have been successful in deleting the PTHrP gene during lactation.

We are currently examining the effects of deleting the PTHrP gene on maternal calcium metabolism. Data thus far demonstrate that the mammary gland structure is normal and that the mice suckle their young successfully, indicating that PTHrP is not necessary for lactation per se. However, our preliminary analysis suggests that the Cre-lox mice have lower levels of circulating PTHrP, lower rates of bone turnover, and higher bone mass. These data, if they are confirmed, would suggest that some PTHrP from the mammary gland does get secreted into the circulation and does participate in the mobilization of skeletal calcium stores in the lactating mothers. An additional finding from these studies is that pups drinking milk without PTHrP appear to have a higher total ash calcium content. Ongoing studies, using these mice will help to elucidate the exact role of PTHrP in maternal and neonatal calcium metabolism during lactation.

Finally, we have also become interested in the role of the calcium sensing receptor (CaR) in the mammary gland. Recent studies have demonstrated that, just like PTHrP, the CaR becomes expressed on mammary epithelial cells specifically during lactation. We have shown that activation of the CaR decreases expression of the PTHrP gene and secretion of PTHrP into media or milk in cultured mammary epithelial cells and in intact mammary glands in lactating mice. Therefore, the lactating mammary gland is a calcium sensing organ and PTHrP secretion by the mammary gland is regulated by systemic calcium levels. If PTHrP does participate in the regulation of bone turnover during lactation, this might constitute a feedback loop whereby, calcium delivery to the gland for milk production is monitored and bone resorption adjusted by altering the amount of PTHrP secreted into the systemic circulation.