Although downstream effects of IFN-γ such as regulation of cellular proliferation, differentiation and apoptosis have been investigated in numerous systems, few studies address the significant effect of this cytokine on mammary epithelial cells under normal or neoplastic conditions (Harvat and Jetten, 1996
; Grunberg et al., 2000
). In addition, the presence of IFN-γ receptors on HMEpCs has not been previously reported. Our studies indicate that HMEpCs possess functional receptor I and II, and receptor engagement results in activating the JAK pathway, leading to phosphorylation of Stat-1 and downstream effects of IFN-γ such as reduced proliferation and loss of epithelial morphology. By utilizing polarized HMEpCs we observed disruption of cell polarity (at IFN-γ concentrations >1ng/ml) and breach in cell-cell contacts confirmed by disruption of the tight junction protein ZO-1. Disassembly of the tight junctions and abrogation of polarized structures are critical steps in normal tissue remodeling and pathogen-induced disruption of epithelial barriers (Ivanov et al., 2004
). Such a process is observed in milk stasis (or mastitis) and could constitute a signal for initiation of mammary gland involution (Shamay et al., 2002
Our studies indicate that IFN-γ decreases the pH of the vacuolar system in HMEpCs, thus altering secretion, processing and enzymatic activity of the aspartyl endopeptidase CatD. Increased proteolytic activity of CatD associated with changes in vacuolar size and pH could reflect the requirement for increased intercellular proteolysis, a process intrinsic to autophagy which is known to occur during the mammary remodeling process (Motyl et al., 2006
; Zarzyńska et al., 2007
). Indeed, IFN-γ induced concentration dependent increases in mRNA and protein expression of an autophagic marker beclin 1(Levine and Yuan, 2005
), concurrent with the appearance of autophagosomes (some containing organelles) was detected by TEM in HMEpCs. To our knowledge this is the first report of INF-γ induction of the autophagic process in mammary epithelial cells and could have important implications in mammary tissue homeostasis -- under normal and pathological conditions.
The effect of INF-γ on secretion of Maspin (and CatD) might reflect INF-γ induced changes in vacuolar biogenesis. Maspin is a member of clad 5B serpins and possesses a facultative secretion signal in its N-terminal region which determines it’s bi-topological (inter- and extra-cellular) distribution (Pemberton et al., 1997
). Reduced vacuolar pH could promote Maspin’s Golgi to plasma membrane transport. This premise is indeed plausible given that inhibition of vATPase is shown to impede the intra-Golgi or Golgi to plasma membrane trafficking of other serpins (Yilla et al., 1993
). It is noteworthy that in polarized HMEpCs, secretion of Maspin and CatD is mostly at the apical surface. IFN-γ treatment, apart from its reciprocal effect on Maspin and CatD secretion, instigates a concentration dependent basolateral release of Maspin with limited effect on apical release of CatD. This altered localized secretion could affect multiple biological functions of mammary tissue including differentiation, cell proliferation and extracellular matrix proteolysis. Studies (Laurent-Matha et al., 2005
, Vetvicka et al., 1994
) indicate that CatD acts as a mitogen via autocrine and paracrine effects, thus, reduced CatD secretion could be a rate limiting factor in cell proliferation. In addition, based on our previous findings (Khalkhali-Ellis and Hendrix 2007
), secreted Maspin could incorporate into the extracellular matrix and regulate extracellular proteolysis and/or cell differentiation --processes fundamental to mammary gland remodeling. The actual occurrence of these events during mammary tissue remodeling is currently under investigation in our developmental mouse model.
Interestingly, the majority of breast cancer cell lines are refractory to INF-γ [(Harvat and Jetten, 1996
), Z-Khalkhali-Ellis, unpublished observation]. This is further supported by our finding of minimal expression of IFN-γ Rs in breast cancer cell lines tested and the failure of IFN-γ to inhibit proliferation, vacuolar acidification and CatD processing in these cell lines. By utilizing LysoSensor we observed far fewer and more basic vacuolar networks in MCF-7 and MDA-MB-231 breast cancer cells compared to HMEpCs. This is in agreement with the reported observation that cancer cells have a diminished ability to acidify their lysosome/endosomes (Kokkonen et al., 2004
). Of the two cancer cell lines tested, MCF-7 cells could form polarized structures, yet in contrast to HMEpCs these were impervious to the effect of IFN-γ. These cancer cell lines are devoid of Maspin, and IFN-γ treatment did not alter their Maspin expression. However, Maspin transfection of these cell lines rendered them more responsive to INF-γ. Specifically, polarized Maspin transfected MCF-7 displayed disrupted tight junctions (similar to those observed in HMEpCs) and a modest increase in beclin-1 when treated with 1ng/ml INF-γ. MDA-MB-231 and its Maspin transfected counterpart failed to polarize under the experimental conditions employed. The differential response of MCF-7 and MDA-MB-231 breast cancer cell lines to INF-γ is unclear and might be the consequence of their tumorigenic potential and differentiation status, which could also reflect the sensitivity of their Maspin transfected counterparts. Our findings define Maspin as a critical component of the INF-γ signaling pathway in mammary epithelial cells and illuminate (at least in part) why breast cancer cells are refractory to IFN-γ. The mechanism by which Maspin sensitizes these cancer cells to this cytokine is currently under investigation in our laboratory and has significant therapeutic implications.
In conclusion, this study provides evidence that IFN-γ receptors are expressed in mammary epithelial cells, and that receptor engagement by IFN-γ regulates vacuolar pH, CatD and Maspin expression. This is achieved by increasing the vacuolar membrane associated V0 sector of vATPase, leading to decreased vacuolar pH and increased CatD activity concomitant with the induction of autophagy, mostly through activation of JAK pathways. Novel aspects of IFN-γ’s effects combined with the ability of Maspin to partially sensitize breast cancer cells to this cytokine might lead to identifying mechanisms which directly activate the antitumor effects of IFN-γ while avoiding its adverse systemic effects.