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We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter choler...
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... [1251]EGF binding was used as the (Table 1). However, when cells were treated with PRL for 30 min there was no significant change in receptor number, but a decrease in the affinity of the high affinity class of receptor from 0.1 nM to 0.5 nM was noted (Table 2). These studies were also performed in the constant presence of 100 ng/ml PRL. ...
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... Most of the previously conducted studies have examined the potential role of growth factors secreted by the mammary epithelium and stroma during normal and pathological changes occurring in the mammary glands of humans and certain mammals. In mice, the majority of literature reports deal with the efficacy of EGF and its receptors (EGFR/erbB-1) (Taketani and Oka 1983a,b; Snedeker et al. 1991; Fenton and Sheffield 1993; Schroeder and Lee 1998; Luetteke et al. 1999; Sebastian et al. 1998). In humans, the majority of studies focus on the role of EGF, VEGF, and their receptors in breast cancer (Klijn et al. 1992; Cunha 1994; Hovey et al. 2001; Rajkumar 2001; Nishimura et al. 2002; Rossiter et al. 2007). ...
... A, alveolus; D, ducts; S, stroma; BV, blood vessel; arrows, positive alveolar and ductal epithelial cells; arrowheads, positive stromal cells. erbBs, VEGI, VEGF, and its receptors in the rat mammary glandexpression in the ductal and alveolar mammary epithelial cells were variable, in compliance with other studies conducted in pregnant rats (Fenton and Sheffield 1993; Sheffield 1998; Wiesen et al. 1999; Schroeder et al. 2001; Stern 2008). In contrast to the reports of Fenton and Sheffield (1993), but in parallel to the observations of Edery et al. (1985), we detected that even though erbB-1 expression was intensified in the ductal and alveolar mammary epithelial cells, particularly at the beginning and mid-stage of pregnancy, its occurrence was delayed to late pregnancy. ...
... erbBs, VEGI, VEGF, and its receptors in the rat mammary glandexpression in the ductal and alveolar mammary epithelial cells were variable, in compliance with other studies conducted in pregnant rats (Fenton and Sheffield 1993; Sheffield 1998; Wiesen et al. 1999; Schroeder et al. 2001; Stern 2008). In contrast to the reports of Fenton and Sheffield (1993), but in parallel to the observations of Edery et al. (1985), we detected that even though erbB-1 expression was intensified in the ductal and alveolar mammary epithelial cells, particularly at the beginning and mid-stage of pregnancy, its occurrence was delayed to late pregnancy. The intensities of staining for the first two trimesters of pregnancy were statistically insignificant ( p . ...
We identified the localization and distribution of cell-specific epidermal growth factor receptors (EGFRs: erbB-1, erbB-2, erbB-3, erbB-4), vascular endothelial growth factor (VEGF), VEGF receptors [VEGFRs: VEGF-R1 (flt-1), VEGF-R2 (flk-1/KDR), VEGF-R3 (flt-4)], vascular endothelial growth inhibitor (VEGI), and estrogen receptor (ER), and determined whether or not these growth factors in rat mammary glands are functional. Thirty-five adult female Spraque-Dawley rats were randomly divided into five groups, each of which were at the 7th, 14th, and 21st day of pregnancy; 7th day post-delivery; and 7th day after weaning. It was determined that erbB, VEGF and its receptors, VEGI, and ER stained at different intensities. Intense staining was observed, in particular, in erbB receptors during pregnancy and involution, and also in VEGF and its receptors during lactation, while ER stained during the last periods of pregnancy and lactation. In conclusion, the expression of erbB, VEGF and its receptors, and ER were determined at varying intensities at different sites of the mammary gland during pregnancy, lactation, and involution periods.
... However, not all interactions between PRL and EGF family members are positive. Some studies of murine cell lines in vitro demonstrate inhibitory interactions between PRL and EGF (Fenton and Sheffield, 1993;Horsch et al., 2001). The relationship of these observations to the other models is unclear. ...
The contributions of prolactin (PRL) to breast cancer are becoming increasingly recognized. To better understand the role for PRL in this disease, its interactions with other oncogenic growth factors and hormones must be characterized. Here, we review our current understanding of PRL crosstalk with other mammary oncogenic factors, including estrogen, epidermal growth factor (EGF) family members, and insulin-like growth factor-I (IGF-I). The ability of PRL to potentiate the actions of these targets of highly successful endocrine and molecular therapies suggests that PRL and/or its receptor (PRLR) may be an attractive therapeutic target(s). We discuss the potential benefit of PRL/PRLR-targeted therapy in combination with established therapies and implications for de novo and acquired resistance to treatment.
... Although signal transduction in response to PRL and ErbB4 have been studied, relatively little is known about how their respective signaling pathways engage to exert a complex biological outcome like differentiation of the mammary epithelium. Recent studies suggest that PRL and GH may participate in cross talk with EGFR and ErbB2 (22)(23)(24)(51)(52)(53)(54)(55)(56). GH causes EGFR phosphorylation on both tyrosines and threonines in a context-specific manner, resulting in enhanced ERK activation and decreased EGFR degradation as well as enhanced or decreased EGFR signaling, depending on the cellular context (21-24, 52, 54). ...
Differentiation of mammary epithelium in vivo requires signaling through prolactin and ErbB4/HER4-dependent mechanisms. Although stimulation of either the prolactin receptor or ErbB4/HER4 results in activation of the transcription factor signal transducer and activator of transcription 5A (STAT5A) and induction of lactogenic differentiation, how these pathways intersect is unknown. We show herein that prolactin signaling in breast cells cooperates with and is substantially enhanced by the receptor tyrosine kinase ErbB4/HER4. Prolactin and the ErbB4/HER4 ligand heparin-binding epidermal growth factor each induced STAT5A tyrosine phosphorylation and nuclear translocation; each pathway required the intracellular tyrosine kinase Janus kinase 2 (JAK2). We found that full prolactin-mediated STAT5A activation and binding to the endogenous beta-casein promoter required ErbB4/HER4 but did not require ErbB1/epidermal growth factor receptor. For example, prolactin-induced STAT5A activity was markedly diminished in cells overexpressing kinase inactive HER4, in cells transfected with small interfering RNAs to specifically knock down endogenous ErbB4/HER4 expression and in cells treated with a small molecule inhibitor that targets ErbB4 kinase. Interestingly, prolactin caused ErbB4/HER4 tyrosine phosphorylation in a JAK2 kinase-dependent manner. Finally, prolactin receptor, ErbB4/HER4, and JAK2 were coimmunoprecipitated from prolactin-treated but not untreated cells. These results suggest that prolactin signaling engages the ErbB4 pathway via JAK2 and that ErbB4 provides an important component of STAT5A-dependent lactogenic differentiation; this pathway integration may help explain the similar deficit in mammary development observed in gene-targeted mice deficient in prolactin receptor, JAK2, ErbB4, or STAT5A.
... Some studies with stimuli other than PMA have further linked such PKC-dependent EGFR phosphorylation to functional consequences. Sheffield and colleagues studied the effects of prolactin on EGFR function in normal murine mammary epithelial cells70717273. In those cells, prolactin caused substantial PKC-dependent EGFR threonine phosphorylation (although the residue was not mapped) and subsequent desensitization of EGF-induced EGFR tyrosine kinase activity. ...
Epidermal growth factor (EGF) signaling is critical in normal and aberrant cellular behavior. Extracellular signal-regulated kinase (ERK) mediates important downstream aspects of EGF signaling. Additionally, EGFR undergoes MEK1-dependent ERK consensus site phosphorylation in response to EGF or cytokines such as growth hormone (GH) and prolactin (PRL). GH- or PRL-induced EGFR phosphorylation alters subsequent EGF-induced EGFR downregulation and signal characteristics in an ERK-dependent fashion. We now use reconstitution to study mutation of the sole EGFR ERK phosphorylation consensus residue, (669)T. CHO-GHR cells, which lack EGFR and express GHR, were stably transfected to express human wild-type or T669A ((669)T changed to alanine) EGFRs at similar abundance. Treatment of cells with GH or EGF caused phosphorylation of WT, but not T669A EGFR, in an ERK activity-dependent fashion that was detected with an antibody that recognizes phosphorylation of ERK consensus sites, indicating that (669)T is required for this phosphorylation. Notably, EGF-induced downregulation of EGFR abundance was much more rapid in cells expressing EGFR T669A vs. WT EGFR. Further, pretreatment with the MEK1/ERK inhibitor PD98059 enhanced EGF-induced EGFR loss in cells expressing WT EGFR, but not EGFR T669A, suggesting that the ERK-dependent effects on EGFR downregulation required phosphorylation of (669)T. In signaling experiments, EGFR T669A displayed enhanced acute (15 min) EGFR tyrosine phosphorylation (reflecting EGFR kinase activity) compared to WT EGFR. Further, acute EGF-induced ubiquitination of WT EGFR was markedly enhanced by PD98059 pretreatment and was increased in EGFR T669A-expressing cells independent of PD98059. These signaling data suggest that ERK-mediated (669)T phosphorylation negatively modulates EGF-induced EGFR kinase activity. We furthered these investigations using a human fibrosarcoma cell line that endogenously expresses EGFR and ErbB-2 and also harbors an activating Ras mutation. In these cells, EGFR was constitutively detected with the ERK consensus site phosphorylation-specific antibody and EGF-induced EGFR downregulation was modest, but was substantially enhanced by pretreatment with MEK1/ERK inhibitor. Collectively, these data indicate that ERK activity, by phosphorylation of a threonine residue in the EGFR juxtamembrane cytoplasmic domain, modulates EGFR trafficking and signaling.
... Effects of PRL on EGF signaling have been studied by Sheffield et al. in normal mammary epithelial cells. In these studies, PRL induced threonine phosphorylation of the EGFR (Fenton and Sheffield, 1993;Johnson et al., 1996;Quijano and Sheffield, 1998). Whereas the sites of phosphorylation were not mapped, pharmacologic data suggested that it might be dependent on protein kinase C (PKC) activation. ...
Prolactin (PRL) is a polypeptide hormone produced by the anterior pituitary gland and other sites that acts both systemically and locally to cause lactation and other biological effects by interacting with the PRL receptor, a Janus kinase (JAK)2-coupled cytokine receptor family member, and activating downstream signal pathways. Recent evidence suggests PRL is a player in the pathogenesis and progression of breast cancer. Epidermal growth factor (EGF) also has effects on breast tissue, working through its receptors, epidermal growth factor receptor (EGFR) and ErbB-2 (c-neu, HER2), both intrinsic tyrosine kinase growth factor receptors. EGFR promotes pubertal breast ductal morphogenesis in mice, and both EGFR and ErbB-2 are relevant in pathogenesis and behavior of breast and other human cancers. Previous studies showed that PRL and EGF synergize to enhance motility in the human breast cancer cell line, T47D. In this study, we explored crosstalk between the PRL and EGF signaling pathways in T47D cells, with an ultimate aim of understanding how these two important factors might work together in vivo to affect breast cancer behavior. Both PRL and EGF caused robust signaling in T47D cells; PRL acutely activated JAK2, signal transducer and activator of transcription-5 (STAT5), and extracellular signal-regulated kinase-1 and -2 (ERK1 and ERK2), whereas EGF caused EGFR activation and consequent src homology collagen (SHC) activation and ERK activation. Notably, PRL also caused phosphorylation of the EGFR and ErbB-2 at sites detected by PTP101, an antibody that recognizes threonine phosphorylation at consensus motifs for ERK-induced phosphorylation. PRL-induced PTP101-reactive phosphorylation was prevented by pretreatment with PD98059, an ERK pathway inhibitor. Furthermore, PRL synergized with EGF in activating SHC and ERK and transactivating a luciferase reporter driven by c-fos gene enhancer elements, suggesting that PRL allowed markedly enhanced EGF signaling. This was accompanied by substantial inhibition of EGF-induced EGFR downregulation when PRL and EGF cotreatment was compared to EGF treatment alone. This effect of PRL was abrogated by ERK pathway inhibitor pretreatment. Our data suggest that PRL synergistically augments EGF signaling in T47D breast cancer cells at least in part by lessening EGF-induced EGFR downregulation and that this effect requires PRL-induced ERK activity and threonine phosphorylation of EGFR.
... Recently, the SOCS (suppressor of cytokine signaling) gene family was identified and they function by negatively regulating the Jak/Stat pathway at the level of activation [86]. Finally, another emerging field in PRLR signaling is the occurrence of cross talk with members of other receptor families, such as tyrosine kinases [87,88] or nuclear receptors [89]. ...
... Finally, another emerging field in PRLR signaling is the occurrence of cross talk with members of other receptor families, such as tyrosine kinases (41,42) or nuclear receptors (43). Interactions of activated Stats with the latter obviously represents a possible molecular mechanism underlying the integrated regulation of multiple hormone-dependent functions known to involve PRL and, for example, sex steroids. ...
Prolactin (PRL) is a paradoxical hormone. Historically known as the pituitary hormone of lactation, it has had attributed to it more than 300 separate actions, which can be correlated to the quasi-ubiquitous distribution of its receptor. Meanwhile, PRL-related knockout models have mainly highlighted its irreplaceable role in functions of lactation and reproduction, which suggests that most of its other reported target tissues are presumably modulated by, rather than strictly dependent on, PRL. The multiplicity of PRL actions in animals is in direct opposition to the paucity of arguments that suggest its involvement in human pathophysiology other than effects on reproduction. Although many experimental data argue for a role of PRL in the progression of some tumors, such as breast and prostate cancers, drugs lowering circulating PRL levels are ineffective. This observation opens new avenues for research into the understanding of whether local production of PRL is involved in tumor growth and, if so, how extrapituitary PRL synthesis is regulated. Finally, the physiological relevance of PRL variants, such as the antiangiogenic 16K-like PRL fragments, needs to be elucidated. This review is aimed at critically discussing how these recent findings have renewed the manner in which PRL should be considered as a multifunctional hormone.
... In mammary tissue, prolactin stimulates the transcription of proteins required for lactation. Although over 100 biological targets of prolactin have been reported [10,12,23,42,48], the significant downstream actions of stress-released prolactin are not known. One potential function of stress-released prolactin is its Values represent the mean of six rats per group S.E.M. ability to promote the immune response and thereby mitigate the immune suppressive effects of glucocorticoids, which are also increased by stress [1,11,42]. ...
... One potential function of stress-released prolactin is its Values represent the mean of six rats per group S.E.M. ability to promote the immune response and thereby mitigate the immune suppressive effects of glucocorticoids, which are also increased by stress [1,11,42]. Prolactin has been shown to alter the transcription of many enzymes in different tissues and some of these effects may prove to play a role in either stress-induced pathophysiology or in stress resistance mechanisms [23,48]. ...
Our laboratory is investigating the effects of chronic stress on physiological, endocrine and behavioral measures, in order to elucidate the neuronal substrates for the pathophysiological consequences of stress in humans. In these studies, we have employed a rodent model of sustained stress in which rats are exposed to around-the-clock intermittent footshock that can be avoided or escaped by rats in the controllable stress group, but not by rats in the uncontrollable stress group. Each rat in the uncontrollable stress group is paired (yoked) to a rat in the controllable stress group such that the controllable stress group rat avoids or escapes shock for both rats. A third group of rats receives no shock (controls). We have previously reported that in male rats, plasma prolactin levels were elevated after 3 days of stress in both stress groups. In the present experiments we determined whether the increases in plasma prolactin were correlated with increases in anterior pituitary prolactin mRNA. In addition, we measured hormones and mRNA at three time points and we examined these responses in female as well as male rats. Adult male and female Sprague-Dawley rats were exposed to chronic stress for 1, 3 or 14 days. In unstressed control rats, levels of anterior pituitary prolactin mRNA were fivefold higher in female as compared to male rats. However, stress increased levels of anterior pituitary prolactin mRNA over baseline in both genders. After 1 day of stress, anterior pituitary prolactin mRNA levels increased in male and female rats belonging to both stress groups, with no significant difference seen between the uncontrollable vs. controllable stress groups. After 3 days of stress, anterior pituitary prolactin mRNA levels were even more elevated, and rats in the uncontrollable stress group had higher anterior pituitary prolactin mRNA levels than those in the controllable stress group. After 14 days of stress, there were no significant differences in control and stressed groups with respect to anterior pituitary prolactin mRNA. These data suggest that chronic sustained stress increases the synthesis of anterior pituitary prolactin mRNA during the first days of stress, and that levels return to prestress values sometime between 3 and 14 days of stress.
... Cells grown to approximately 80% confluency were lysed with lysis buffer (50 mM HEPES, pH 7.0 containing 30 mM sodium pyrophosphate, 10 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100,m 0.1% BSA, 1 mM sodium orthovanadate and 1 mM PMSF) and centrifuged (15,000 g for 15 minutes). Protein content of the supernatant was determined by BCA assay, equalized among cell lines and c-src immunoprecipitated from equal protein amounts essentially as previously described (21) (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)) (1 mM for standard assays). Reactions were started by adding 5 /M of a 0.5 mM y 32 P-ATP solution (approximately 1 Ci/mmole, Dupont, Boston, MA). ...
... using anti-c-src (UBI) and agarose conjuated protein A and G (Santa Cruz Biotechnology, Santa Cruz, CA). Beads were washed 4 times with lysis buffer and c-src activity determined by incubating immunoprecipitated enzyme in 15 p\ of assay buffer (200 mM HEPES, pH 7.0 containing 125 mM MgCl 2 , 25 mM MnCl 2 , and 0.25 mM sodium orthovanadate) with or without substrate peptide ([lys19 ]cdc2(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)) or control pep- ...
... This observation would be in accordance with previous reports (12) that EGF receptor numbers in mouse mammary tissue decline during late pregnancy and lactation and that EGF receptor is lower in lactating bovine mammary tissue than in nonlactating tissue (15). PRL has also been shown to decrease EGF receptor numbers and mRNA in murine mammary epithelial cells after approximately 18 h of treatment (36). ...
Mammary tissue from midpregnant heifers was cultured with epidermal growth factor (EGF) or transforming growth factor alpha for 1-3 days. After 1 day, 10 nM EGF or transforming growth factor alpha doubled DNA synthesis, whereas lower concentrations (0.1 or 1 nM) increased DNA synthesis 2- to 3-fold after 2-3 days in culture. In other studies, bovine mammary tissue was transplanted to ovariectomized athymic mice and treated for 10 days with saline, estradiol (1 microg/day), progesterone (1 mg/day), or estradiol + progesterone. Subsequent explant culture of the bovine tissue indicated that estradiol + progesterone augmented the ability of EGF to stimulate DNA synthesis. The increased response to EGF was associated with increased EGF binding and with increased EGF-induced tyrosine kinase that paralleled the increased EGF binding. In other studies, athymic mice bearing xenografted bovine mammary tissue were primed for 10 days with estradiol and progesterone, followed by 2-day treatment with saline (control), hydrocortisone (200 microg/day), PRL (1 mg/day), or hydrocortisone + PRL. Hydrocortisone and PRL alone decreased, and PRL + hydrocortisone eliminated, EGF-induced DNA synthesis. EGF receptor content was unaffected by hydrocortisone but was reduced by PRL or hydrocortisone + PRL. Furthermore, the ability of EGF to induce tyrosine kinase activity was decreased by PRL and by hydrocortisone + PRL. The decreased kinase activity was greater than the decrease in receptor binding, suggesting a specific modulation of EGF receptor kinase activity in response to lactogenic hormones.
