MCB Accepted Manuscript Posted Online 23 February 2015 Mol. Cell. Biol. doi:10.1128/MCB.00136-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Hasegawa et al., 2015 1
Epithelial Xbp1 is Required for Cellular Proliferation and Differentiation
2
During Mammary Gland Development
3 4
Daisuke Hasegawa1,2, Veronica Calvo3, Alvaro Avivar-Valderas3, Abigale Lade1,
5
Hsin-I Chou1, Youngmin A. Lee1, Eduardo F Farias3, Julio A. Aguirre-Ghiso3, Scott L. Friedman1*
6 7
1
Division of Liver Diseases, Icahn School of Medicine at Mount Sinai. New York, New York, USA
8
2
Division of Gastroenterology and Hepatology, St. Marianna University School of Medicine, Kawasaki,
9
Japan
10
3
11
Cancer Institute, Icahn School of Medicine at Mount Sinai New York , USA
Division of Hematology and Oncology, Department of Medicine, Department of Otolaryngology, Tisch
12 13
Running Title: Role of epithelial Xbp1 in mammary gland in vivo
14
Keywords: Xbp1; Prolactin receptor; Stat5 phosphorylation; mammary epithelial cells; ER stress; UPR
15
*Correspondence address:
16
Scott L. Friedman, MD
17
Division of Liver Diseases, 1425 Madison Ave, Room 1170C, Box 1123, Icahn School of Medicine at
18
Mount Sinai, New York, New York, USA 10029
19
E-mail: [email protected]
20
Word count: Abstract: 195 words; Materials and Methods: 9686 characters; Abstract, Introduction,
21
Results, Discussion, and Figure legends, 1 Table and 9 figures: 33786 characters (excluding spaces)
22 1
Hasegawa et al., 2015 23
Abstract
24
Xbp1, a key mediator of the unfolded protein response (UPR), is activated by IRE1-mediated splicing,
25
which results in a frameshift to encode a protein with transcriptional activity. However, the direct
26
function of Xbp1 in epithelial cells during mammary gland development is unknown. Here we report
27
that loss of Xbp1 in mammary epithelium through targeted deletion leads to poor branching
28
morphogenesis, impaired terminal end bud formation and spontaneous stromal fibrosis during the adult
29
virgin period. Additionally, epithelial Xbp1 deletion induces endoplasmic reticulum (ER) stress in the
30
epithelium and dramatically inhibits epithelial proliferation and differentiation during lactation.
31
Synthesis of the milk and its major components α/ß-casein and whey acidic protein (WAP) are
32
significantly reduced due to decreased prolactin receptor (Prlr) and ErbB4 expression in Xbp1-deficient
33
mammary epithelium. Reduction of Prlr and ErbB4 expression and their diminished availability at the
34
cell surface lead to reduced phosphorylated Stat5, an essential regulator of cell proliferation and
35
differentiation during lactation. As a result, lactating mammary glands in these mice produce less milk
36
protein, leading to poor pup growth and postnatal death. These findings suggest that loss of Xbp1
37
induces a terminal UPR which blocks proliferation and differentiation during mammary gland
38
development.
39
2
Hasegawa et al., 2015 40
Introduction
41
The primary function of the mammary gland is to provide nutrition for newborns through
42
production of milk protein and lipids (1). These milk proteins are synthesized in the endoplasmic
43
reticulum (ER) and are secreted in to the mammary duct as classical secretory proteins (2). The
44
mammary gland undergoes dramatic, continual developmental changes throughout adulthood and
45
provides a valuable model through which to track the interplay between the secretory pathway
46
competence and epithelial cell maturation during postnatal development (3). Development of the
47
mammary gland is governed by hormonal stimuli, which includes the prolactin /ErbB4/Stat 5 signaling
48
axis (4-8). During pregnancy the mammary epithelium grows and branches until mid-pregnancy, and
49
differentiates functionally during late pregnancy and the early postpartum period. This epithelial
50
differentiation is accompanied by the expression of milk protein genes, such as whey acidic protein
51
(Wap) and α/ß-casein, and by the production of milk droplets (6).
52
The ER has a crucial role in the quality control during the folding and secretion of secretory
53
proteins. The accumulation of misfolded proteins in the ER provokes ER stress by increasing the
54
demand for energy, chaperones and other proteins that are needed to fold client proteins or to degrade
55
unfoldable secretory cargo. This stress activates a signaling network called the unfolded protein
56
response (UPR). The UPR increases the folding capacity of the secretory pathway through the
57
transcriptional and the up-regulation of ER chaperones and foldases and the ER quality control
58
machinery. Xbp1 is one master regulator of the UPR. It is produced as an RNA that is regulated by
59
Ire1-mediated cytoplasmic splicing of Xbp1 mRNA, resulting in a frameshift that then creates an mRNA
60
that encodes a transcriptionally active protein (spliced or sXbp1). Spliced Xbp1 regulates the
61
transcription of a number of ER quality control genes and is essential for the development, survival and
62
function of intestinal epithelial cells, immune cells, hepatocytes (9) and adipocytes (10) as well as for
63
the development of professional secretory cells such as B-cells, hepatocytes and pancreatic ß cells (11,
64
12).
Two recent studies have characterized the contribution of the UPR to lactation. The first has 3
Hasegawa et al., 2015 65
implicated the PERK arm of the UPR in regulating lipogenesis in mammary epithelium during lactation
66
(13). In the second study, adipocyte Xbp1 was proven essential for activity of the lactating gland, and
67
Xbp1 splicing in adipocytes was induced by the
68
changes in Xbp1 splicing in adipocytes did not alter milk composition, mammary lipogenic activity or
69
function as assessed by Stat5 phosphorylation, expression of prolactin or ErbB4 receptor (10).
70
Accordingly, the contribution of mammary epithelium Xbp1 to breast epithelial cell function and
71
development is unknown.
lactogenic hormone prolactin. However, these
72
We have recently reported that ER stress induces fibrogenic activity in hepatic stellate cells,
73
which are the key fibrogenic cells in the liver, through activation of Ire1α/Xbp1 signaling (14). This
74
observation raised the possibility that there are pathophysiological levels of chronic ER stress associated
75
with tissue fibrosis. During our study to explore the role of Xbp1 in mesenchymal cells using the
76
Cre-LoxP genetic recombination system using hGFAP-Cre (15-18) crossed to Xbp1fl/fl mice we
77
discovered that Xbp1fl/fl;hGFAP-Cre transgenic mice effectively targeted mammary epithelial cells
78
rather than mesenchymal cells. In these animals, we observed that chronic ER stress caused by loss of
79
Xbp1 in mammary epithelial cells induces alteration in the ductal epithelium, with spontaneous stromal
80
fibrosis in mammary glands, and these changes disrupt mammary gland development, with impaired
81
epithelial cell proliferation and differentiation. Thus, serendipitously, the present study reveals a
82
previously unrecognized function for Xbp1 in the regulation of mammary gland development.
83
4
Hasegawa et al., 2015 84
Materials and methods
85
Generation and breeding of Xbp1fl/fl;GFAP-Cre Mice
86
Xbp1fl/fl mice were a gift from Drs. Laurie H. Glimcher (Weill Cornell Medical College). Xbp1fl/fl mice
87
previously described (19) on the C57BL/6 background were crossed with a transgenic FVB line
88
expressing Cre recombinase under the control of the human glial fibrillary acid protein (hGFAP)
89
promoter (GFAP-Cre) (15) to generate Xbp1fl/fl;GFAP-Cre mice. For cell fate studies, hGFAP-cre mice
90
were crossed with B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J reporter mice (20). A breeding
91
strategy was followed that ensured that control Xbp1fl/fl and experimental mice Xbp1fl/fl;GFAP-Cre were
92
always littermates. Xbp1fl/fl and Xbp1fl/fl;GFAP-Cre dams that delivered a litter within 24h of each other
93
were used in cross-fostering experiments. The litters born to Xbp1fl/fl;GFAP-Cre and Xbp1fl/fl dams were
94
swapped as described (21). All experimental mice were anesthetized (ketamine 100 mg/kg, xylazine
95
20mg/kg) and humanely euthanized prior to collection of tissue; conduct of our experiments have
96
complied with the highest international criteria. All studies were approved by the Institutional Animal
97
Care and Use Committee of the Icahn School of Medicine at Mount Sinai and followed the National
98
Institutes of Health guidelines for animal care.
99 100
Antibodies
101
The antibodies used in this study were as follows: anti-GFP antibody (1:1000) (ab6556; Abcam),
102
Anti-K8/18 antibody (1:200) (Progen Biotechnik), anti-αSMA conjugated with Cy3 antibody (1:500)
103
(C6198; Sigma), anti-Xbp1 antibody (1:200) (M-186; Santa Cruz), anti-PDI antibody (#3501), anti-Bip
104
antibody (1:1000) (#3177), anti-CHOP antibody (1:1000) (#2895), anti-IRE1α antibody (1:1000)
105
(#3294) (Cell signaling), anti-GAPDH antibody (1:5000) (ab9482; Abcam), anti-Ki67 antibody(1:2500)
106
(ab15580; Abcam), anti-phospho-histone H3 (Ser10) Antibody (1:300) (#9701; Cell signaling),
107
anti-cleaved caspase-3 (Asp175) Antibody (1:300) (#9661; Cell signaling), anti-phospho-Stat5 (Tyr694)
5
Hasegawa et al., 2015 108
(C71E5) antibody (IHC;1:400 WB; 1:1000) (#9314; Cell signaling), anti-Stat5 antibody (1:1000)
109
(#9363; Cell signaling) and anti-mouse milk specific protein antibody (1:5000) (Accurate Chemical).
110 111
Preparation of tissue protein and immunoblot analysis
112
For total protein extraction, mouse mammary gland tissues and cells were lysed in RIPA buffer [50 mM
113
Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.2% SDS] containing protease and phosphatase inhibitors
114
(Roche). Protein content was measured by Bio-Rad Protein Assay Dye Reagent (500-0006). Proteins
115
were resolved by NuPAGE® 4-12% Bis-Tris Gel (Invitrogen) and transferred to PVDF membranes.
116
Blots were probed with the primary antibody for overnight at 4 °C, washed, and incubated with
117
secondary anti-rabbit or mouse IgG, HRP-linked antibody (#7074 and #7076; Cell signaling) for 1 h at
118
room temperature. Blots were developed using HyGlo Quick Spray Reagent (Denville).
119 120
Genotyping PCR
121
Xbp1fl/fl;hGFAP-Cre and ROSA26mT/mG;hGFAP-Cre mice were genotyped via PCR analysis, as
122
previously described (19). The presence of the floxed Xbp1 allele was detected via PCR using mouse
123
tail DNA as a template. A PCR product specific for the floxed Xbp1 allele was amplified by using the
124
following
125
CAAGGTGGTTCACTGCCTGTAATG 3′ (reverse). The PCR conditions were as follows: 94 °C for 3
126
min, followed by 35 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min (size of
127
PCR product: WT : 141 bp, flox : 183 bp). The presence of the hGFAP-Cre transgene was detected by
128
using
129
5’CAGGGTGTTATAAGCAATCCC 3’ (reverse). The PCR conditions were as follows: 94 °C for 4 min
130
followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1.5 min and 72 °C for 10 min (size of
131
PCR product: 400 bp).
primers:
following
PCR
5′ACTTGCACCAACACTTGCCATTTC
primers:
3’
5’CCTGGAAAATGCTTCTGTCCG
132 6
(forward)
3’
and
(forward)
5’
and
Hasegawa et al., 2015 133
Real-time PCR
134
PCR primers for real-time PCR are summarized in Table S1. Fresh isolated mammary tissues were
135
frozen and stored at -80 °C until specimens were homogenized by bead milling using the TissueLyser
136
LT (Qiagen, Hilden, Germany), which rapidly disrupts up to 12 samples simultaneously via high-speed
137
vertical shaking (oscillation frequency of 50 Hz) with a bead (7 mm) in a sealed tube for 3 min. Then,
138
mammary tissue RNA was extracted using Qiagen mini columns and RNeasy mini kits (Qiagen,
139
Germantown, MD) with an on-column deoxyribonuclease treatment. One microgram of RNA was
140
reverse-transcribed using RT Complete Double PrePrimed Kit (Clontech, Mountain View, CA).
141
FastStart SYBR Green Master (Roche, Indianapolis, IN) was used for polymerase chain reaction.
142
Samples were analyzed in triplicate in Microsoft Excel (Microsoft Corp, Redmond, WA) and
143
normalized to ß-actin expression.
144 145
Whole-mount analysis and histological analysis
146
Whole-mount analysis of mouse mammary glands was performed as described previously (22).
147
Mammary glands were excised and spread on microscope slides. The tissues were fixed in 10% formalin
148
at 4ºC, defatted in Carboy’s fixative, washed in 70% ethanol, hydrated by passing through decreasing
149
ethanol concentration and stained in carmine alum stain [0.2% carmine, 0.5% aluminum potassium
150
sulfate] overnight at room temperature. For histological analysis, mammary gland specimens were fixed
151
overnight in 10% formalin at 4ºC, dehydrated and embedded in paraffin. Tissue blocks were sectioned
152
into 5 micrometer and stained with hematoxylin and eosin (H-E). Sirius Red (Sigma) was used to
153
determine collagen deposition. Sections were stained with Sirius Red solution (saturated picric acid
154
containing 0.1% Direct Red 80 and 0.1% Fast Green) to visualize collagen deposition. Relative fibrosis
155
area was assessed based on 20 fields from sirius red-stained mammary gland sections per animal. Whole
156
field areas were from the mammary luminal area and each field was acquired at 400-fold field
157
magnification and then analyzed using a computerized Bioquant® morphometry system (R & M 7
Hasegawa et al., 2015 158
Biometrics, Nashville, TN, USA). To evaluate the relative fibrosis area, the measured collagen area was
159
divided by the net field area.
160 161
Immunohistochemistry and immunofluorescence
162
Tissues were immediately fixed in 10% neutral buffered formalin for 24 h. After fixation, the tissue was
163
immersed in 70% ethanol until processing. All tissues were processed simultaneously. The fixed tissues
164
were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks, which were cooled
165
before sectioning. The mammary gland tissues were sectioned into 4-µm-thick slices, and the sections
166
were mounted on silane-coated slides. For antigen activation, the slides were incubated in Target
167
Retrieval Solution (DAKO) and heated for 30 min in a microwave oven. The slides were allowed to
168
cool. Then, sections were used for immunohistochemistry with DAKO EnVision detection system using
169
immunoperoxidase method according to the DAKO EnVision kit manual.
170
All immunohistochemistry staining were probed with the primary antibody overnight at 4 °C. The slides
171
were then washed three times in PBS and incubated with secondary anti-rabbit or mouse antibody for
172
1 h at room temperature. For immunofluorescence analysis, Alexa Fluor® 488 Donkey anti-rabbit IgG
173
(H+L) antibody (1:500) (Invitrogen) and Alexa Fluor® 647 Goat Anti-guinea pig IgG (H+L) antibody
174
(1:500) (Life Technologies) were used as secondary antibodies. Isotype control was used to assess
175
non-specific binding. The slides were mounted with DAPI ProLong Gold antifade reagent (P36931, Life
176
Technologies, Carlsbad, CA, USA) and examined in a Zeiss axiophot photomicroscope (Carl Zeiss,
177
Oberkochen, Germany). The number of DAB-positive cells for Ki-67, p-Histone H3 and cleaved
178
caspase 3 were counted in the stained sections at 200 or 400-fold field magnification under a
179
microscope, using 20 randomly selected microscopic fields per section.
180 181
Isolation of mouse mammary epithelial cells
8
Hasegawa et al., 2015 182
Mammary epithelial cells were isolated as previously described, with minor modifications (23). Inguinal
183
glands from ROSA26mT/mG;hGFAP-Cre and Xbp1fl/fl;hGFAP-Cre mice were removed aseptically,
184
minced and digested with collagenase at 37°C for 45 minutes. Digested glands were subsequently
185
centrifuged at 1,000 rpm for 5 minutes, and the fat layer and supernatant removed. The pellet was
186
washed and incubated in red blood cell lysis buffer (Sigma R7757, St. Louis, MO, USA) for 2 minutes
187
before centrifugation. Cells were then plated in DMEM +10% FBS+1% P/S and incubated for 30
188
minutes at 37°C to allow for the selective attachment of fibroblasts. The supernatant was transferred into
189
BSA-coated tubes and washed once before resuspension in MCF10A medium (DMEM/F12
190
supplemented with antibiotics, EGF (20 ng/ml), hydrocortisone (0.5 ug/ml), insulin (10ug/ml), cholera
191
toxin (0.1ug/ml) and 5% heat inactivated horse serum) on a 10 cm dish coated with Matrigel (BD, San
192
Jose, CA, USA).
193 194
Mammary gland transplantation
195
Transplantation experiments were performed as described previously (24). Pubescent three/four-week
196
old donor and host female mice were anesthetized and mid-sagittal and oblique cuts were made in order
197
to expose the 4th inguinal mammary gland. The fat pad in the mammary gland was cleared from the host
198
epithelium by removing the tissue anterior to the lymph node. Epithelial fragments (2 mm3) of the donor
199
mice were transplanted into the remaining cleared fat pad of the host (Xbp1fl/fl;GFAP-Cre pad into
200
Xbp1fl/fl mice and Xbp1fl/fl pad into Xbp1fl/fl;GFAP-Cre mice) and closed. After six weeks, transplanted
201
pregnant females were sacrificed at day 7 of lactation. The transplanted glands were excised and
202
processed for whole-mount analysis as described.
203 204
Evaluation of serum prolactin level
205
Xbp1fl/fl;GFAP-Cre (n=3) and Xbp1fl/fl (n=5) littermate mice were mated after mammary gland
206
transplantation and sacrificed at day 7 of lactation. Blood was collected via inferior vena cava puncture 9
Hasegawa et al., 2015 207
and allowed to clot overnight at 4°C. Samples were centrifuged at 4000 g for 15 min and serum was
208
collected and stored at −80°C. Prolactin concentrations were assessed by prolactin mouse ELISA Kit
209
(Abcam).
210 211
RT-PCR for Xbp1 splicing assay
212
RT-PCR analysis was performed using PCR Master Mix (2X) (Thermo). A PCR product specific for the
213
spliced XBP1 was amplified by using the following primers: 5′ACACGCTTGGGAATGGACAC 3’
214
(forward) and 5’ CCATGGGAAGATGTTCTGGG 3′ (reverse). For spliced Xbp1 and cyclophilin
215
detection by standard RT- PCR, the following program was used: (1) 94°C for 3 min, (2) 30 cycles of
216
94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, (3) 72 °C for 10 min. PCR products were separated by
217
agarose gel electrophoresis to resolve the 171 bp (unspliced) and 145 bp (spliced) amplicons. RT-PCR
218
for cyclophilin mRNA was performed to validate cDNA synthesis as loading control.
219 220
Evaluation of serum cholesterol and triglyceride concentration
221
Quantitative determination of serum cholesterol and triglyceride levels was performed by a kit from
222
(Pointe Scientific, Inc, Canton, MI) using spectrophotometer analysis.
223 224
Statistical analysis
225
Statistical analysis was performed using a commercially available software package (Prism 5.0;
226
Graphpad Software). Data were tested by Student's t-test. Differences were considered statistically
227
significant at p
Hasegawa et al., 2015 1
Epithelial Xbp1 is Required for Cellular Proliferation and Differentiation
2
During Mammary Gland Development
3 4
Daisuke Hasegawa1,2, Veronica Calvo3, Alvaro Avivar-Valderas3, Abigale Lade1,
5
Hsin-I Chou1, Youngmin A. Lee1, Eduardo F Farias3, Julio A. Aguirre-Ghiso3, Scott L. Friedman1*
6 7
1
Division of Liver Diseases, Icahn School of Medicine at Mount Sinai. New York, New York, USA
8
2
Division of Gastroenterology and Hepatology, St. Marianna University School of Medicine, Kawasaki,
9
Japan
10
3
11
Cancer Institute, Icahn School of Medicine at Mount Sinai New York , USA
Division of Hematology and Oncology, Department of Medicine, Department of Otolaryngology, Tisch
12 13
Running Title: Role of epithelial Xbp1 in mammary gland in vivo
14
Keywords: Xbp1; Prolactin receptor; Stat5 phosphorylation; mammary epithelial cells; ER stress; UPR
15
*Correspondence address:
16
Scott L. Friedman, MD
17
Division of Liver Diseases, 1425 Madison Ave, Room 1170C, Box 1123, Icahn School of Medicine at
18
Mount Sinai, New York, New York, USA 10029
19
E-mail: [email protected]
20
Word count: Abstract: 195 words; Materials and Methods: 9686 characters; Abstract, Introduction,
21
Results, Discussion, and Figure legends, 1 Table and 9 figures: 33786 characters (excluding spaces)
22 1
Hasegawa et al., 2015 23
Abstract
24
Xbp1, a key mediator of the unfolded protein response (UPR), is activated by IRE1-mediated splicing,
25
which results in a frameshift to encode a protein with transcriptional activity. However, the direct
26
function of Xbp1 in epithelial cells during mammary gland development is unknown. Here we report
27
that loss of Xbp1 in mammary epithelium through targeted deletion leads to poor branching
28
morphogenesis, impaired terminal end bud formation and spontaneous stromal fibrosis during the adult
29
virgin period. Additionally, epithelial Xbp1 deletion induces endoplasmic reticulum (ER) stress in the
30
epithelium and dramatically inhibits epithelial proliferation and differentiation during lactation.
31
Synthesis of the milk and its major components α/ß-casein and whey acidic protein (WAP) are
32
significantly reduced due to decreased prolactin receptor (Prlr) and ErbB4 expression in Xbp1-deficient
33
mammary epithelium. Reduction of Prlr and ErbB4 expression and their diminished availability at the
34
cell surface lead to reduced phosphorylated Stat5, an essential regulator of cell proliferation and
35
differentiation during lactation. As a result, lactating mammary glands in these mice produce less milk
36
protein, leading to poor pup growth and postnatal death. These findings suggest that loss of Xbp1
37
induces a terminal UPR which blocks proliferation and differentiation during mammary gland
38
development.
39
2
Hasegawa et al., 2015 40
Introduction
41
The primary function of the mammary gland is to provide nutrition for newborns through
42
production of milk protein and lipids (1). These milk proteins are synthesized in the endoplasmic
43
reticulum (ER) and are secreted in to the mammary duct as classical secretory proteins (2). The
44
mammary gland undergoes dramatic, continual developmental changes throughout adulthood and
45
provides a valuable model through which to track the interplay between the secretory pathway
46
competence and epithelial cell maturation during postnatal development (3). Development of the
47
mammary gland is governed by hormonal stimuli, which includes the prolactin /ErbB4/Stat 5 signaling
48
axis (4-8). During pregnancy the mammary epithelium grows and branches until mid-pregnancy, and
49
differentiates functionally during late pregnancy and the early postpartum period. This epithelial
50
differentiation is accompanied by the expression of milk protein genes, such as whey acidic protein
51
(Wap) and α/ß-casein, and by the production of milk droplets (6).
52
The ER has a crucial role in the quality control during the folding and secretion of secretory
53
proteins. The accumulation of misfolded proteins in the ER provokes ER stress by increasing the
54
demand for energy, chaperones and other proteins that are needed to fold client proteins or to degrade
55
unfoldable secretory cargo. This stress activates a signaling network called the unfolded protein
56
response (UPR). The UPR increases the folding capacity of the secretory pathway through the
57
transcriptional and the up-regulation of ER chaperones and foldases and the ER quality control
58
machinery. Xbp1 is one master regulator of the UPR. It is produced as an RNA that is regulated by
59
Ire1-mediated cytoplasmic splicing of Xbp1 mRNA, resulting in a frameshift that then creates an mRNA
60
that encodes a transcriptionally active protein (spliced or sXbp1). Spliced Xbp1 regulates the
61
transcription of a number of ER quality control genes and is essential for the development, survival and
62
function of intestinal epithelial cells, immune cells, hepatocytes (9) and adipocytes (10) as well as for
63
the development of professional secretory cells such as B-cells, hepatocytes and pancreatic ß cells (11,
64
12).
Two recent studies have characterized the contribution of the UPR to lactation. The first has 3
Hasegawa et al., 2015 65
implicated the PERK arm of the UPR in regulating lipogenesis in mammary epithelium during lactation
66
(13). In the second study, adipocyte Xbp1 was proven essential for activity of the lactating gland, and
67
Xbp1 splicing in adipocytes was induced by the
68
changes in Xbp1 splicing in adipocytes did not alter milk composition, mammary lipogenic activity or
69
function as assessed by Stat5 phosphorylation, expression of prolactin or ErbB4 receptor (10).
70
Accordingly, the contribution of mammary epithelium Xbp1 to breast epithelial cell function and
71
development is unknown.
lactogenic hormone prolactin. However, these
72
We have recently reported that ER stress induces fibrogenic activity in hepatic stellate cells,
73
which are the key fibrogenic cells in the liver, through activation of Ire1α/Xbp1 signaling (14). This
74
observation raised the possibility that there are pathophysiological levels of chronic ER stress associated
75
with tissue fibrosis. During our study to explore the role of Xbp1 in mesenchymal cells using the
76
Cre-LoxP genetic recombination system using hGFAP-Cre (15-18) crossed to Xbp1fl/fl mice we
77
discovered that Xbp1fl/fl;hGFAP-Cre transgenic mice effectively targeted mammary epithelial cells
78
rather than mesenchymal cells. In these animals, we observed that chronic ER stress caused by loss of
79
Xbp1 in mammary epithelial cells induces alteration in the ductal epithelium, with spontaneous stromal
80
fibrosis in mammary glands, and these changes disrupt mammary gland development, with impaired
81
epithelial cell proliferation and differentiation. Thus, serendipitously, the present study reveals a
82
previously unrecognized function for Xbp1 in the regulation of mammary gland development.
83
4
Hasegawa et al., 2015 84
Materials and methods
85
Generation and breeding of Xbp1fl/fl;GFAP-Cre Mice
86
Xbp1fl/fl mice were a gift from Drs. Laurie H. Glimcher (Weill Cornell Medical College). Xbp1fl/fl mice
87
previously described (19) on the C57BL/6 background were crossed with a transgenic FVB line
88
expressing Cre recombinase under the control of the human glial fibrillary acid protein (hGFAP)
89
promoter (GFAP-Cre) (15) to generate Xbp1fl/fl;GFAP-Cre mice. For cell fate studies, hGFAP-cre mice
90
were crossed with B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J reporter mice (20). A breeding
91
strategy was followed that ensured that control Xbp1fl/fl and experimental mice Xbp1fl/fl;GFAP-Cre were
92
always littermates. Xbp1fl/fl and Xbp1fl/fl;GFAP-Cre dams that delivered a litter within 24h of each other
93
were used in cross-fostering experiments. The litters born to Xbp1fl/fl;GFAP-Cre and Xbp1fl/fl dams were
94
swapped as described (21). All experimental mice were anesthetized (ketamine 100 mg/kg, xylazine
95
20mg/kg) and humanely euthanized prior to collection of tissue; conduct of our experiments have
96
complied with the highest international criteria. All studies were approved by the Institutional Animal
97
Care and Use Committee of the Icahn School of Medicine at Mount Sinai and followed the National
98
Institutes of Health guidelines for animal care.
99 100
Antibodies
101
The antibodies used in this study were as follows: anti-GFP antibody (1:1000) (ab6556; Abcam),
102
Anti-K8/18 antibody (1:200) (Progen Biotechnik), anti-αSMA conjugated with Cy3 antibody (1:500)
103
(C6198; Sigma), anti-Xbp1 antibody (1:200) (M-186; Santa Cruz), anti-PDI antibody (#3501), anti-Bip
104
antibody (1:1000) (#3177), anti-CHOP antibody (1:1000) (#2895), anti-IRE1α antibody (1:1000)
105
(#3294) (Cell signaling), anti-GAPDH antibody (1:5000) (ab9482; Abcam), anti-Ki67 antibody(1:2500)
106
(ab15580; Abcam), anti-phospho-histone H3 (Ser10) Antibody (1:300) (#9701; Cell signaling),
107
anti-cleaved caspase-3 (Asp175) Antibody (1:300) (#9661; Cell signaling), anti-phospho-Stat5 (Tyr694)
5
Hasegawa et al., 2015 108
(C71E5) antibody (IHC;1:400 WB; 1:1000) (#9314; Cell signaling), anti-Stat5 antibody (1:1000)
109
(#9363; Cell signaling) and anti-mouse milk specific protein antibody (1:5000) (Accurate Chemical).
110 111
Preparation of tissue protein and immunoblot analysis
112
For total protein extraction, mouse mammary gland tissues and cells were lysed in RIPA buffer [50 mM
113
Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.2% SDS] containing protease and phosphatase inhibitors
114
(Roche). Protein content was measured by Bio-Rad Protein Assay Dye Reagent (500-0006). Proteins
115
were resolved by NuPAGE® 4-12% Bis-Tris Gel (Invitrogen) and transferred to PVDF membranes.
116
Blots were probed with the primary antibody for overnight at 4 °C, washed, and incubated with
117
secondary anti-rabbit or mouse IgG, HRP-linked antibody (#7074 and #7076; Cell signaling) for 1 h at
118
room temperature. Blots were developed using HyGlo Quick Spray Reagent (Denville).
119 120
Genotyping PCR
121
Xbp1fl/fl;hGFAP-Cre and ROSA26mT/mG;hGFAP-Cre mice were genotyped via PCR analysis, as
122
previously described (19). The presence of the floxed Xbp1 allele was detected via PCR using mouse
123
tail DNA as a template. A PCR product specific for the floxed Xbp1 allele was amplified by using the
124
following
125
CAAGGTGGTTCACTGCCTGTAATG 3′ (reverse). The PCR conditions were as follows: 94 °C for 3
126
min, followed by 35 cycles at 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and 72 °C for 5 min (size of
127
PCR product: WT : 141 bp, flox : 183 bp). The presence of the hGFAP-Cre transgene was detected by
128
using
129
5’CAGGGTGTTATAAGCAATCCC 3’ (reverse). The PCR conditions were as follows: 94 °C for 4 min
130
followed by 40 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1.5 min and 72 °C for 10 min (size of
131
PCR product: 400 bp).
primers:
following
PCR
5′ACTTGCACCAACACTTGCCATTTC
primers:
3’
5’CCTGGAAAATGCTTCTGTCCG
132 6
(forward)
3’
and
(forward)
5’
and
Hasegawa et al., 2015 133
Real-time PCR
134
PCR primers for real-time PCR are summarized in Table S1. Fresh isolated mammary tissues were
135
frozen and stored at -80 °C until specimens were homogenized by bead milling using the TissueLyser
136
LT (Qiagen, Hilden, Germany), which rapidly disrupts up to 12 samples simultaneously via high-speed
137
vertical shaking (oscillation frequency of 50 Hz) with a bead (7 mm) in a sealed tube for 3 min. Then,
138
mammary tissue RNA was extracted using Qiagen mini columns and RNeasy mini kits (Qiagen,
139
Germantown, MD) with an on-column deoxyribonuclease treatment. One microgram of RNA was
140
reverse-transcribed using RT Complete Double PrePrimed Kit (Clontech, Mountain View, CA).
141
FastStart SYBR Green Master (Roche, Indianapolis, IN) was used for polymerase chain reaction.
142
Samples were analyzed in triplicate in Microsoft Excel (Microsoft Corp, Redmond, WA) and
143
normalized to ß-actin expression.
144 145
Whole-mount analysis and histological analysis
146
Whole-mount analysis of mouse mammary glands was performed as described previously (22).
147
Mammary glands were excised and spread on microscope slides. The tissues were fixed in 10% formalin
148
at 4ºC, defatted in Carboy’s fixative, washed in 70% ethanol, hydrated by passing through decreasing
149
ethanol concentration and stained in carmine alum stain [0.2% carmine, 0.5% aluminum potassium
150
sulfate] overnight at room temperature. For histological analysis, mammary gland specimens were fixed
151
overnight in 10% formalin at 4ºC, dehydrated and embedded in paraffin. Tissue blocks were sectioned
152
into 5 micrometer and stained with hematoxylin and eosin (H-E). Sirius Red (Sigma) was used to
153
determine collagen deposition. Sections were stained with Sirius Red solution (saturated picric acid
154
containing 0.1% Direct Red 80 and 0.1% Fast Green) to visualize collagen deposition. Relative fibrosis
155
area was assessed based on 20 fields from sirius red-stained mammary gland sections per animal. Whole
156
field areas were from the mammary luminal area and each field was acquired at 400-fold field
157
magnification and then analyzed using a computerized Bioquant® morphometry system (R & M 7
Hasegawa et al., 2015 158
Biometrics, Nashville, TN, USA). To evaluate the relative fibrosis area, the measured collagen area was
159
divided by the net field area.
160 161
Immunohistochemistry and immunofluorescence
162
Tissues were immediately fixed in 10% neutral buffered formalin for 24 h. After fixation, the tissue was
163
immersed in 70% ethanol until processing. All tissues were processed simultaneously. The fixed tissues
164
were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks, which were cooled
165
before sectioning. The mammary gland tissues were sectioned into 4-µm-thick slices, and the sections
166
were mounted on silane-coated slides. For antigen activation, the slides were incubated in Target
167
Retrieval Solution (DAKO) and heated for 30 min in a microwave oven. The slides were allowed to
168
cool. Then, sections were used for immunohistochemistry with DAKO EnVision detection system using
169
immunoperoxidase method according to the DAKO EnVision kit manual.
170
All immunohistochemistry staining were probed with the primary antibody overnight at 4 °C. The slides
171
were then washed three times in PBS and incubated with secondary anti-rabbit or mouse antibody for
172
1 h at room temperature. For immunofluorescence analysis, Alexa Fluor® 488 Donkey anti-rabbit IgG
173
(H+L) antibody (1:500) (Invitrogen) and Alexa Fluor® 647 Goat Anti-guinea pig IgG (H+L) antibody
174
(1:500) (Life Technologies) were used as secondary antibodies. Isotype control was used to assess
175
non-specific binding. The slides were mounted with DAPI ProLong Gold antifade reagent (P36931, Life
176
Technologies, Carlsbad, CA, USA) and examined in a Zeiss axiophot photomicroscope (Carl Zeiss,
177
Oberkochen, Germany). The number of DAB-positive cells for Ki-67, p-Histone H3 and cleaved
178
caspase 3 were counted in the stained sections at 200 or 400-fold field magnification under a
179
microscope, using 20 randomly selected microscopic fields per section.
180 181
Isolation of mouse mammary epithelial cells
8
Hasegawa et al., 2015 182
Mammary epithelial cells were isolated as previously described, with minor modifications (23). Inguinal
183
glands from ROSA26mT/mG;hGFAP-Cre and Xbp1fl/fl;hGFAP-Cre mice were removed aseptically,
184
minced and digested with collagenase at 37°C for 45 minutes. Digested glands were subsequently
185
centrifuged at 1,000 rpm for 5 minutes, and the fat layer and supernatant removed. The pellet was
186
washed and incubated in red blood cell lysis buffer (Sigma R7757, St. Louis, MO, USA) for 2 minutes
187
before centrifugation. Cells were then plated in DMEM +10% FBS+1% P/S and incubated for 30
188
minutes at 37°C to allow for the selective attachment of fibroblasts. The supernatant was transferred into
189
BSA-coated tubes and washed once before resuspension in MCF10A medium (DMEM/F12
190
supplemented with antibiotics, EGF (20 ng/ml), hydrocortisone (0.5 ug/ml), insulin (10ug/ml), cholera
191
toxin (0.1ug/ml) and 5% heat inactivated horse serum) on a 10 cm dish coated with Matrigel (BD, San
192
Jose, CA, USA).
193 194
Mammary gland transplantation
195
Transplantation experiments were performed as described previously (24). Pubescent three/four-week
196
old donor and host female mice were anesthetized and mid-sagittal and oblique cuts were made in order
197
to expose the 4th inguinal mammary gland. The fat pad in the mammary gland was cleared from the host
198
epithelium by removing the tissue anterior to the lymph node. Epithelial fragments (2 mm3) of the donor
199
mice were transplanted into the remaining cleared fat pad of the host (Xbp1fl/fl;GFAP-Cre pad into
200
Xbp1fl/fl mice and Xbp1fl/fl pad into Xbp1fl/fl;GFAP-Cre mice) and closed. After six weeks, transplanted
201
pregnant females were sacrificed at day 7 of lactation. The transplanted glands were excised and
202
processed for whole-mount analysis as described.
203 204
Evaluation of serum prolactin level
205
Xbp1fl/fl;GFAP-Cre (n=3) and Xbp1fl/fl (n=5) littermate mice were mated after mammary gland
206
transplantation and sacrificed at day 7 of lactation. Blood was collected via inferior vena cava puncture 9
Hasegawa et al., 2015 207
and allowed to clot overnight at 4°C. Samples were centrifuged at 4000 g for 15 min and serum was
208
collected and stored at −80°C. Prolactin concentrations were assessed by prolactin mouse ELISA Kit
209
(Abcam).
210 211
RT-PCR for Xbp1 splicing assay
212
RT-PCR analysis was performed using PCR Master Mix (2X) (Thermo). A PCR product specific for the
213
spliced XBP1 was amplified by using the following primers: 5′ACACGCTTGGGAATGGACAC 3’
214
(forward) and 5’ CCATGGGAAGATGTTCTGGG 3′ (reverse). For spliced Xbp1 and cyclophilin
215
detection by standard RT- PCR, the following program was used: (1) 94°C for 3 min, (2) 30 cycles of
216
94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, (3) 72 °C for 10 min. PCR products were separated by
217
agarose gel electrophoresis to resolve the 171 bp (unspliced) and 145 bp (spliced) amplicons. RT-PCR
218
for cyclophilin mRNA was performed to validate cDNA synthesis as loading control.
219 220
Evaluation of serum cholesterol and triglyceride concentration
221
Quantitative determination of serum cholesterol and triglyceride levels was performed by a kit from
222
(Pointe Scientific, Inc, Canton, MI) using spectrophotometer analysis.
223 224
Statistical analysis
225
Statistical analysis was performed using a commercially available software package (Prism 5.0;
226
Graphpad Software). Data were tested by Student's t-test. Differences were considered statistically
227
significant at p
