We observed that p190A, like p190B, is expressed throughout the developing mammary gland and that its function is required for proper mammary gland morphogenesis. Furthermore, our results suggest that p190A plays a role in mediating cell adhesion within the TEB and that complete loss of p190A leads to altered mammary gland differentiation. These results suggest that p190A has similar, but distinct roles from p190B within the mammary gland, as it does in the developing neural system (Matheson et al., 2006
). For example, loss of one allele of p190B delayed mammary gland outgrowth between 4 and 6 weeks, while heterozygosity for p190A did not significantly delay development past 4 weeks. The increased expression of p190B within the cap cell layer may account for the difference in the outgrowth potential, as the TEB is the driving force of ductal outgrowth due to the high proliferative capacity of the cap cell layer (Chakravarty et al., 2003
; Humphreys et al., 1997
Interestingly, haploinsufficiency for p190A disrupts the TEB architecture resulting in adhesion defects within the cap cell layer and body cell layer. Reduced levels of the axon guidance molecule neogenin, that is known to play an important role in cap and body cell adhesion within TEBs (Srinivasan et al., 2003
), may in part underlie the adhesion defect. Other genes involved in axonal guidance, slit2/robo1, have also been shown to play a role in adhesion with the TEB and ducts of the developing mammary gland (Srinivasan et al., 2003
; Strickland et al., 2006
). These proteins are members of the plexin family (Hinck, 2004
). Interestingly, p190A deficient fibroblasts are blocked or greatly impaired with respect to the typical functional activities mediated by plexins, including cell collapse and inhibition of integrin-based adhesion. Re-expression of wildtype p190A, but not a mutant form specifically lacking RhoGAP activity, rescued plexin-mediated functions in these cells (Barberis et al., 2005
). Both members of the p190 family have been shown to accumulate at sites of integrin crosslinking, while only p190A has been shown to function downstream of cadherin engagement suggesting a novel mechanism through which p190A may be mediating this adhesion defect (Burbelo et al., 1995
; Noren et al., 2003
; Sharma, 1998
). Further studies are required to determine the mechanisms by which p190A regulates adhesion within the mammary gland.
P190A knockout mice die perinatally or just after birth due to defects in forebrain midline structures (Brouns et al., 2000
). At E18.5 the p190A-deficient embryonic mammary glands are indistinguishable from those in wildtype littermates. We, therefore, used embryonic mammary gland transplantation techniques to examine the effects of complete loss of p190A on mammary gland development. The use of both EF and WG transplants allowed us to investigate the role of p190A in both the epithelium and stroma. These results revealed p190A was important in both compartments as EF and WG p190A-deficient transplants demonstrated reduced outgrowth potential, and WG deficient transplants also displayed reduced transplantation efficiency. Interestingly, the outgrowth potential is greater in the p190A-deficient glands than observed previously in the p190B-deficient glands (Chakravarty et al., 2003
), suggesting that p190B may compensate for some functions of p190A, while p190A appears not to compensate for the loss of p190B. While these proteins normally may have some overlapping functions, a compensatory up regulation of p190B in the absence of p190A has not been previously observed (Brouns et al., 2000
Histological examination of p190A-deficient EF transplants revealed reduced stromal condensation around the ductal epithelium as well as the persistence of TEB-like structures. This is in contrast to our studies of p190B overexpression in the mammary gland where we have observed an increase in stromal condensation (Vargo-Gogola et al., 2006
). This is of interest because activity of Rho kinase (ROK), a downstream effector, is required for breast epithelial cells to sense the rigidity of their environment and down regulate Rho activity to differentiate (Wozniak et al., 2003
). Thus, the lack of stromal condensation in the p190A-deficient glands may lead to aberrant differentiation.
In addition to changes in matrix deposition, a significant increase in the number of steroid receptor positive cells as well as the level of expression was detected by immunostaining. At 10–12 weeks of age, the steroid receptor expression changes from a pattern where it is present in a high percentage of cells to a restricted pattern (Silberstein et al., 1996
). This change in receptor expression pattern correlates with a reduction in proliferation of the ductal epithelium (Clarke et al., 1997
). No difference in the proliferative capacity of the mature ductal epithelium within the wildtype and p190A-deficient transplants was observed in our studies, although this analysis could only be conducted on those samples in which outgrowth had occurred. The approximately 50% reduction in percentage of fat pad filled in the EF transplants suggests that the p190A deficient mammary epithelium do grow at a reduced rate. In addition, Rho GTPases and their regulators have been implicated in estrogen dependent proliferation, invasion, and transcriptional activation in ER positive breast cancer cell lines (Barone et al.
; Su et al., 2001
; Walker et al.
; Xie and Haslam, 2008
). It is intriguing to speculate that Rho signaling proteins, including p190A, may also regulate ER dependent cellular processes in the developing mammary gland.
Further examination of the p190A-deficient transplants also points to p190A’s putative role as a tumor suppressor. P190A-deficient transplants have a reduced myoepithelial cell layer. Myoepithelial cells secrete fibronectin, collagen, nidogen, and laminin to form the basement membrane. The reduction of myoepithelial cells may contribute to the reduced collagen present in the stroma. Disruption of the myoepithelial cell layer is also seen following p190B overexpression, which led to hyperplastic lesions during pregnancy (Vargo-Gogola et al., 2006
Previous studies have shown that overexpression of p190A resulted in abnormal positioning of the cleavage furrow during cytokinesis (Su et al., 2003
), and that gene silencing of p190A was also detrimental to cell cycle progression. These results, in combination with data presented here implicating p190A in differentiation of the mammary epithelium, may suggest a role for p190A in symmetric versus asymmetric division of mammary progenitor cells. A defect in symmetric versus asymmetric division may also potentially underlie the aberrant differentiation of the mammary epithelium seen in the p190A-deficient outgrowths.
These studies have revealed a critical role for p190A in both the epithelial and stromal compartments in the developing mammary gland. Interestingly, three studies have implicated p190A as a tumor suppressor (Tikoo et al., 2000
; Wang et al., 1997
; Wolf et al., 2003
). Our studies investigating p190B loss and gain of function in the MMTV-Neu induced mammary tumor model suggest that p190B functions as an oncogene during the stochastic process of mammary tumorigenesis in vivo
(Heckman-Stoddard et al., 2009
; McHenry PR, 2010
) Future studies will be aimed at investigating the role of p190A during mammary tumorigenesis.
P190A RhoGAP is expressed in the epithelium and stroma of the developing embryonic mammary bud and postnatal mammary gland
P190A is required in both the epithelium and stroma for mammary ductal development.
P190A deficient terminal end buds display defects in adhesion between the cap and body cell compartments.
The proportion of ERα and PR positive mammary epithelial cells is increased in the p190A−/− ductal epithelium, suggesting that p190A may affect mammary epithelial cell differentiation.