IFN-gamma regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells

Abstract

In this study we examined the ability of interferon-gamma (IFN-gamma) to regulate mammary epithelial cell growth and gene expression, with particular emphasis on two genes: Maspin (a member of serine protease inhibitor superfamily), and the lysosomal aspartyl endopeptidase cathepsin D (CatD). The protein products of these genes are critically involved in regulation of multitude of biological functions in different stages of mammary tissue development and remodeling. In addition, the expression of Maspin is down-regulated in primary breast cancer and is lost in metastatic disease, while CatD is excessively produced and aberrantly secreted by breast cancer cells. We report that IFN-gamma receptors are expressed in mammary epithelial cells, and receptor engagement by IFN-gamma transduces the IFN-gamma signal via Stat-1 resulting in decreased vacuolar pH. This change in vacuolar pH alters CatD protein processing and secretion concurrent with increased Maspin secretion. In addition, IFN-gamma exerts a suppressive effect on cell growth and proliferation, and induces morphological changes in mammary epithelial cells. Our studies also reveal that breast cancer cells, which are devoid of Maspin, are refractory to IFN-gamma with respect to changes in vacuolar pH and CatD. However, Maspin transfection of breast cancer cells partially sensitizes the cells to IFN-gamma's effect, thus providing new therapeutic implications.

Fig. 1

(A) Western blot analysis of post-nuclear cytosolic extract from control and IFN-γ treated HMEpCs demonstrates the presence of IFN-γ R I (multiple bands of∼80-95 kDa), and RII (∼38kDa major band and a smaller molecular mass of ∼33-35kDa, most likely a cleavage product of the 38kDa form). Changes in epithelial cell morphology in response to IFN-γ was assessed using monolayer cultures either on plastic (non-polarized) (B), or on transwell culture dishes (polarized) (C-I). Immunofluorescence staining of polarized HMEpCs with antibody to Muc-1 reveals the presence of Muc-1 (green fluorescence) at the apical surface determined by Z stacking of the confocal image (F). CatD staining (red) is included to indicate changes in CatD distribution and intensity in control versus treated HMEpCs. Tight junctions are highlighted by ZO-1 staining (red fluorescence) of untreated HMEpCs (G), and their disruption (arrows) following IFN-γ treatment is depicted in (H-I). Original magnifications are 63x. C, F, and G depict control untreated and D, E, H, and I represent IFN-γ treated HMEpCs.

Fig. 2

Western blot analysis of CM (A) and cytosolic extracts (B) from control and IFN-γ treated HMEpCs indicate concentration-dependent decreases in secreted CatD (sCatD) and intermediate CatD (ICatD) with minimal changes in 34kDa mature CatD (MCatD) (A&B). For comparative purpose, Western blot analysis of early endosomal antigen 1 (EEA1) and Lamp-1 are included (C) to indicate minimal effect on other endosomal/lysosomal enzymes. Note the inverse correlation between secreted Maspin (sMaspin) and secreted CatD (sCatD) in response to IFN-γ treatment (A). The secretion of Maspin and CatD in polarized HMEpCs are mostly at the apical surface and IFN-γ treatment instigates basolateral release of Maspin without affecting the CatD apical secretion (D). Contrary to IFN-γ, chloroquine (CQ) and bafilomycin A (Baf) treatment of HMEpCs result in accumulation of ICatD and decreased MCatD (E). The SDS-PAGE was performed on a 10% (A, B & C) and 12.5% (D) resolving gel. For loading control either ß-actin or a picture of the stained post transfer gel is included.

Fig.3

Examination of vacuolar pH with LysoSensor prior to (A) and after IFN-γ treatment (B&C), 1 and 5 ng/ml IFN-γ, respectively indicates reduced pH resulting in increased yellow emission(arrow). The effect of IFN-γ on pH and CatD processing is abrogated using the JAK inhibitor p6 (D, magnification: 40x). PCR analysis of mRNA from the control and IFN-γ treated HMEpCs reveals a concentration dependent increase in the “E” subunit of v₀ATPase (E). This is confirmed by Western blot analysis of the cytosolic fraction of control and IFN-γ treated HMEpCs probed for the 100kDa subunit “a” of the V₀ sector of vATPase (F). The band corresponding to ∼50kDa is most likely a degradation product of the 100 kDa subunit. Changes in cell associated CatD enzymatic activity in response to IFN-γ is depicted in (G). For loading control a picture of the stained post transfer gel is included in (F).

Fig. 4

Concentration-dependent changes in beclin 1 mRNA (A) and protein expression (B) in HMEpCs in response to IFN-γ. TEM of control (C) and IFN-γ treated HMEpCs (D) indicate the presence of membrane bounded vacuoles (open arrow) in the HMEpCs treated with 1ng/ml IFN-γ. Autophagic vacuoles were identified by their characteristic double membrane (arrow, E). Note the numerous vacuoles (most probably secretory vacuoles) in both treated and control cells. Direct magnification is 10,000 (C&D) and 75,000 (E).

Fig.5

Western blot analysis of cytosolic fractions from HMEpCs (prior to and after exposure to IFN-γ) depicts elevated Stat-1 expression and phosphorylation in response to increasing concentrations of IFN-γ (A). Utilizing the JAK inhibitor P6 mitigates the effect of IFN-γ on Stat-1 (B) and normalizes CatD processing (C). A 10% SDA-PAGE was used in these experiments and a picture of the stained gel is included to confirm equal loading. ICatD: intermediate CatD, MCatD: mature CatD.

Fig. 6

The vacuolar pH of MCF-7 and MDA-MB-231 breast cancer cell lines and their Maspin transfected counterparts prior to and after IFN-γ treatment was examined using LysoSensor Blue/Yellow DND160. Note the higher vacuolar pH in cancer cell lines compared to that of HMEpCs (depicted in Fig. 3 A-D of the manuscript).

Fig.7

Western blot analysis of post-nuclear cytosolic extracts from control and IFN-γ treated MCF-7 and MDA-MB-231 breast cancer cells indicate minimal changes in intermediate CatD (ICatD) (except a slight increase in MDA-MB-231 at low IFN-γ concentrations) in both cell lines (A&C). Maspin transfection of MCF-7 and MDA-MB-231 cells sensitizes them to IFN-γ effect (B&D). 10 % SDS-PAGE gels were used in these experiments. MCF-7 breast cancer cells form polarized structures determined by immunostaining with the polarity indicator Muc-1 (green) and Z stacking by confocal microscopy (E). The aggregates of MCF-7 formed in culture are also polarized (arrow heads). Red fluorescence depicts CatD and the nuclei are counterstained with Dapi. Examination of tight junction integrity with an antibody against ZO-1 indicates minimal effect of IFN-γ on tight junction assembly in MCF-7 cells (F&G), while tight junction integrity was abrogated (arrows) in IFN-γ treated Maspin transfected MCF-7 (H&I). The green fluorescence (Alexa 488) depicts EEA-1 staining. Bar depicts 10μm and the original magnification is 63×10 with 2.5xscan zoom.

Fig.8

MDA-MB-231 breast cancer cells fail to form polarized structures when cultured in transwell tissue culture dishes; Maspin transfection did not alter the response. Muc-1 (green fluorescence), CatD (red fluorescence, rhodamine) and the nucleus (blue, Dapi) immunostaining of control and IFN-γ treated (1 and 5ng/ml) cells are depicted in A-C, and Z stacking is shown in D. E represents control MDA-MB-231 immunostaining with anti-EEA-1 (green) and ZO-1 (red) to demonstrate the absence of tight junctions.