Current dogma suggests that a full term pregnancy results in permanent changes in key proliferative (IGF-I & TGF β) and tumors suppressor (p53) genes in the mammary gland protecting it from carcinogenesis (13
). Although these alterations undoubtedly contribute to the protective effect, more recent evidence suggests that other systemic changes may independently modulate the sensitivity of the mammary glands to carcinogenesis. Specifically, rodent studies have shown that mammary tumorigenesis is decreased when either p53-null mammary epithelial cells (17
) or AMV rat mammary epithelial cells exposed to the chemical carcinogen N-methyl-N-nitrosourea (MNU) (16
) are transplanted into estrogen/progesterone pre-treated or parous rodents. These studies suggest that pregnancy, at least in part, changes the systemic environment, altering endocrine hormones known to regulate mammary gland signaling, thus reducing the susceptibility of the mammary gland to tumors.
In the current study we show that a single full term pregnancy results in a decrease in basal circulating levels of GH in two different strains of rats. This confirms and extends a previous observation by Thordarson et al.
which showed that a single time point measurement of serum GH in 120 day old parous SD females was lower compared to AMV (11
). Given the episodic secretion of GH in the female rat (37
), our study represents a more accurate depiction of circulating GH levels and shows that the parity-induced change are due to an overall reduction in GH secretion and not to alterations in pulse frequency or pulse amplitude. Importantly, the alteration in circulating GH levels was associated with decreased p-Jak2 and p-Stat5A signaling within the mammary gland, suggesting that a global suppression of GH secretion may in part reduce the mammary gland sensitivity to GH. Based on other studies indicating a reduction in carcinogen-induced mammary tumorigenesis in rats with low levels of GH, and increased tumorigenesis following GH replacement (24
), our data would support the concept that pregnancy reduction of GH may in part be responsible for parity-induced protection from breast cancer.
Significant data implicates GH and IGF-I action in both mammary gland development and tumorigenesis. The most compelling evidence linking GH with mammary carcinogenesis comes from studies using the SD Spontaneous Dwarf Rat (SDR), which has a mutation in the GHRH gene resulting in non-detectable circulating GH and subsequently low IGF-I levels (24
). Recently, Shen et al.
showed that SDR females treated with the chemical carcinogen MNU do not develop mammary tumors, however rat or bovine GH replacement in these animals initiated mammary tumorigenesis comparable to MNU-treated controls (27
). Similar results were shown in Lewis SDRs, which have a less severe GH deficiency, but are still resistant to chemically-induced mammary tumorigenesis (43
). Furthermore, transgenic overexpression of GH in mice results in spontaneous mammary tumors (21
), whereas growth of human breast cancer cells is severely retarded in mice lacking GH secretion compared to wild type controls (44
). In addition, GH administration to aging primates results in mammary gland hyperplasia (45
). However, relatively little data exist implicating GH-induced breast cancer in humans. A recent review of numerous clinical studies suggested that height positively correlates with an increase in breast cancer incidence (46
). More importantly, GH-R expression is increased in neoplastic breast tumors (30
) and GH is also expressed in breast cancers (47
In this study we showed that pregnancy in the rat resulted in a moderate reduction (26 % in WF and 37% in SD) in circulating GH levels. While GH is known to be a major modulator of circulating IGF-I levels, the reduction in GH was not associated with decreased IGF-I. In fact we actually observed a small increase in circulating IGF-I similar to that previously reported by Thordarson et al.
). Although unexpected, human obesity studies have shown that GH suppression can exist without subsequent reduction in circulating IGF-I levels (49
). It is possible that the reduction didn’t reach a threshold necessary for altering IGF-I levels, or that IGF-I secretion is mainly modulated by alterations in amplitude and frequency of GH release, both of which we found to be unchanged by IGF-I (data not shown). Our result could be explained, in part, by examining the paracrine feedback loops in the liver responsible for IGF-I production and secretion. We showed that the moderate reduction (26 % in WF and 37% in SD) in GH due to parity was not sufficient enough to alter liver ALS mRNA expression levels, a protein which has been shown to be independently regulated by GH (53
). Furthermore, parity had no effect on IGFBP-3 protein concentrations in serum (data not shown); thus, suggesting that paracrine feedback loops regulating IGF-I secretion in the liver remained enacted. Supporting this are previous studies in mice showing that both ALS and IGFBP-3 produced in the liver are required for the stability of IGF-I (55
). Overexpression of IGFBP-3 results in increased plasma IGF-I (56
) and knockout of ALS results in decreased levels of circulating IGF-I and IGFBP-3 (55
). Therefore a single full term pregnancy may not alter IGF-I or it could be that that assessing GH levels 28 days after weaning was premature, not allowing for the body to conform to new homeostatic conditions, and subsequent analyses of parous animals at a later date may reveal more dramatic changes in IGF-I signaling due to chronic suppression of GH. In addition, extending our current animal model to include multiple pregnancies may also positively reflect a previous report showing that women having 4 or more pregnancies have significantly reduced circulating levels of IGF-I (34
), but a single full term pregnancy only resulted in a 12ng/ml non-significant difference vs. nulliparous women, which translated to our current study, is similar.
While we found that decreased circulating GH didn’t affect circulating IGF-I levels, we did still detect a decrease in liver GH-R mRNA in parous animals. Previous studies have shown that hepatic GH-R mRNA is decreased during pregnancy and this coincides with a significant decrease in circulating levels of IGF-I (57
). Hepatic GH-R levels remain suppressed throughout lactation, however plasma IGF-I levels return to normal when compared to early pregnancy levels (57
). We didn’t measure pre-pregnancy levels of liver GH-R or serum IGF-I, however, it is possible that the parity-induced decrease in liver GH-R mRNA levels may reflect a persistent decrease that first occurred during pregnancy. Furthermore, the normalization of IGF-I levels that occurs during lactation are presumably carried through to involution and parity and may in part explain the paradox of decreased liver GH-R mRNA but unaltered circulating IGF-I level.
The mechanism whereby a full term pregnancy regulates the episodic release of GH from the anterior pituitary is largely unknown. In WF rats, pituitary GH mRNA expression and serum GH is increased during mid-pregnancy, but by day 8 of lactation both mRNA expression and serum levels are similar to early pregnancy (57
). At the end of gestation, the cellular composition of the anterior pituitary changes dramatically due to the increase in prolactin producing lactotrophic cells (necessary for lactation); thus reducing the availability of GH producing somatotrophic cells (57
). Therefore the cellular balance in the pituitary may never fully be restored. However, permanent pregnancy-induced alterations in the pituitary are unlikely since we observed no change in pituitary GH mRNA levels in parous WF rats compared to AMV. This would suggest that after pregnancy GH transcriptional activity returns to normal as well as the ratio of somatotrophs to lactotrophs in the anterior pituitary. Therefore, the more likely scenario is that the parity-induced reduction in serum GH levels is due to changes in regulatory inputs responsible for GH secretion. GH production and secretion from the anterior pituitary is primarily regulated by hypophysiotropic hormones GH-releasing hormone (GHRH) and somatostatin (inhibitory) as well as negatively regulated by circulating IGF-I (for review see ref. (58
). Given that we found no change in circulating levels in IGF-I it is unlikely that IGF-I is inhibiting GH release. Furthermore, GHRH directly controls the production and release of GH during pregnancy (59
). Therefore, pregnancy could directly alter hypothalamic secretion of GHRH and/or somatostatin resulting in the observed decreased circulating levels of GH without directly altering GH transcription. How this is occurring is difficult to speculate. GHRH and somatostatin are highly regulated by hypothalamic neurotransmitters and neuropepites which integrate to determine the pulsatile secretion of GH (60
), thus presenting an arduous challenge in deciphering the mechanic regulation of GHRH induced GH release in the parous animal. Additionally, metabolic peptides such as ghrelin (62
) and glucocorticoids (63
) can directly affect GH secretion from the pituitary and it might be that permanently pregnancy alters metabolic rates that account for the change in GH release.
Global gene expression studies have shown alteration in key pathways critical for mammary gland development and homeostasis including GH-R, IGF-I, amphiregulin, and TGF-β (15
). We found that amphiregulin mRNA was lower in parous mammary gland (data not shown), however, parity didn’t alter levels of GH-R or IGF-I mRNA. This is in contrast to recent microarray data showing that GH-R and IGF-I mRNA expression levels are lower in rat mammary glands due to parity compared to age matched AMVs (42
). However, the conflicting result may simply be explained by the analytical methods used in the two studies given we measured mRNA expression by Q-PCR. However, Thordarson et. al
also showed that pregnancy reduced serum GH levels but did not alter GH-R mRNA expression in the mammary glands of SD rats compared to AMVs (11
Thus far, the effect of reducing mRNA levels of growth factor signaling components in the mammary gland hasn’t been correlated with altered activation status of proteins in the same pathways. To this end, we examined signaling in the parous mammary gland compared to AMV and found that pregnancy reduces mammary gland levels of p-Jak2, p-Stat5A, and p-Akt; all key regulators of mammary cell proliferation and differentiation (64
). Jak2 and Stat5A are essential downstream elements in GH-R signaling transduction, suggesting that the parous mammary gland maybe insensitive to GH action. This is an important observation, given that several studies have identified that alterations to the Jak2/Stat5 signaling pathway in the mammary gland leads to cancer (67
In conclusion, we demonstrated that a single full term pregnancy significantly decreased circulating basal levels of GH, a hormone known to regulate mammary gland development (18
) and shown to play a role in mammary tumorigenesis (21
). This reduction in serum GH was not sufficient enough to alter circulating IGF-I levels. However, the parity-induced reduction in serum GH downregulated key canonical GH signaling proteins critical for mammary gland epithelial proliferation. Our results suggest that pregnancy reduces circulating levels of GH, possibly by altering hypothalamic regulatory mechanisms. This correlates with reduced sensitivity to GH in mammary stromal cells, resulting in stromal-epithelial paracrine communication pathways that may lead to altered mammary gland function making it less susceptible to tumorigenesis. Therefore, GH may play a role in pregnancy induced protection from breast cancer.