2. ERBB1–4 receptors and their EGF-like ligands are present in the
uroepithelium of the normal urinary bladder and urothelial cancer. Sev-
eral studies have linked enhanced expression of ERBB1/EGFR with high
tumor stage, fast progression, and poor clinical outcome in bladder car-
cinoma (Chow et al., 1997; Rotterud et al., 2005; Kramer et al., 2007;
Kim et al., 2005; el-Marjou et al., 2000; Naik et al., 2011). Notably, up-
regulation of BTC was reported in chemically induced mouse bladder
cancer (el-Marjou et al., 2000).
In the present study, we assessed the expression of BTC and its re-
ceptors EGFR/ERBB1 and ERBB4 in the urinary bladder of BTC transgenic
mice (Schneider et al., 2005), mice carrying the antimorphic Wa5 allele
(Lee et al., 2004), and in BTC transgenic/Wa5 mouse models (Schneider
et al., 2009). BTC was detected in stromal microvascular structures and
in the apical umbrella cell lining of the uroepithelium. At 5 months of
age, BTC and BTC/Wa5 mice showed urothelial hyperplasia suggesting
that BTC signaling was not solely dependent on ERBB1/EGFR signaling.
BTC was not detected in urine. However, systemic BTC over-expression
coincided with significantly lower urinary content of major urinary pro-
teins (MUPs) exclusively in female BTC transgenic mice.
2. Materials and methods
2.1. Generation and genotyping of BTC transgenic mice
BTC transgenic (tg) mice were maintained in the FVB/N background
and genotyped as described previously (Schneider et al., 2005). Waved5
(Wa5) mice expressing a dominant kinase-dead EGFR mutant were
provided by the Medical Research Council (Oxfordshire, UK) via
Dr. David Threadgill (University of North Carolina) (Lee et al., 2004).
All animal experiments were approved by the institutional animal
care committee and carried out in accordance with the German Animal
Welfare Act.
2.2. Tissue preparation and immunohistochemistry
Mouse urinary bladders were fixed in 4% buffered formalin and
paraffin-embedded. For immunohistochemistry, tissue sections (5 μm
thick) were deparaffinized by immersion in xylene for twice 10 min
followed by a descending ethanol series and equilibration in tris-
buffered saline (TBS) at pH 7.6. Epitope retrieval was performed by boil-
ing in 10 mM citrate buffer at pH 6.0 for 30 min and activity of endoge-
nous peroxidase was quenched with 3% peroxide in methanol for
20 min, with three TBS washings after each step. For the detection of
BTC, tissue sections were incubated with 1:200 dilution of polyclonal
goat anti-mouse BTC antibodies (AF1025; R&D Systems, Minneapolis,
MN, USA) in TBS with 0.1% Tween 20 (TBST) in a humid chamber at
4 °C overnight. After three washing steps, a 1:200 dilution of biotinylat-
ed rabbit anti-goat IgG antibodies (PK-6105; Vector Laboratories,
Burlingame, CA, USA) was applied for 60 min at room temperature
(RT). For immunodetection of EGFR/ERBB1, ERBB4, and CD44v6, a
1:100 dilution of polyclonal rabbit antisera to EGFR (2232; Cell Signaling
Technology, USA), ERBB4 (SC-283; Santa Cruz Biotechnologies, CA,
USA), and CD44v6 (AB2080; Millipore, Temecula, CA, USA) were used
with biotinylated goat anti-rabbit IgG secondary antibodies (BA-1000;
Vector Laboratories). For the detection of Ki67 proliferation marker,
a 1:100 dilution of monoclonal rat anti-Ki67 antibody (M7249;
DakoCytomation, Glostrup, Denmark) was used with biotinylated rab-
bit anti-goat IgG secondary antibodies (PK-6105; Vector Laboratories).
Sections were incubated for 30 min in avidin–biotin complex (Vector
Laboratories,) and 2× DAB substrate (Thermo Scientific, Waltham, MA
USA) for specific immunodetection of the antibodies used. Nonspecific
binding sites were blocked for 1 h at RT with 5% normal rabbit or goat
serum (Sigma, Saint Louis, MO, USA) of the same species as the
secondary antibody and non-immune IgG at the same concentration
as the primary antiserum was used as a negative control. Specific immu-
nostaining was developed using the DAB kit (Pierce, IL, USA). Tissue
sections were counterstained with hematoxylin and embedded prior
to bright field imaging with a Zeiss M2 microscope (Zeiss, Jena,
Germany). Images were captured and processed with an AxioCam cam-
era and Zeiss Axiovision software, respectively.
2.3. Morphometric analysis
Morphometry was performed on images at ×200 magnification of
H&E stained and ERBB4 immunostained urinary bladders using the
AxioVision and Zen software system (Zeiss). Mice with the four geno-
types were divided into three age groups: 2–4, 5–6, and 8–12 months
(Table 1). The height of the uroepithelium was determined in sections
located approximately medially in non-distended urinary bladders as
determined by microscopic inspection. We measured the shortest dis-
tance from the basal membrane to the lumen along the grain of the tis-
sue and excluded non-perpendicular regions. The average height of the
urinary bladder epithelium was calculated as a mean of at least 500 in-
dividual measurements and up to 2150 measurements for each animal;
exceptions included bladder sections of 2, 5, and 12 month old animals
where only 251, 136, and 386 measurements could be obtained, respec-
tively. The weighted mean and standard deviation for each age group
was calculated and results are presented as dot plots.
2.4. Protein preparation from mouse urine samples
To determine if the increased BTC expression in BTC transgenic mice
resulted in detectable BTC levels and changes of urinary proteins in
urine, we collected urine from three wild type and three tg BTC males
at 2 months of age and four 8 month old female mice of the same geno-
types for protein analysis. For mass spectrometry, urine samples (10 μl)
were diluted with 12.5 μl 1× Laemmli buffer and filled up with 2.5 μl 5×
Laemmli buffer to a final volume of 15 μl. The diluted urine samples
were separated by a 12% SDS-PAGE and stained with Coomassie-
Brilliant-Blue-R (Sigma). Every protein lane of the gel was cut into sev-
eral slices for trypsin digestion. Gel slices were equilibrated twice with
50 mM NH4HCO3 for 10 min and reduced with 45 mM dithiothreitol
for 30 min at 55 °C. Cysteine residues were blocked with 100 mM
iodoacetamide for 30 min at RT, washed twice for 15 min in 50 mM
NH4HCO3, minced and subjected to overnight digestion at 37 °C with
1 μg porcine trypsin (Promega, Madison, WI, USA) per slice. The super-
natant was preserved and peptides were further extracted by additional
washes with 50 mM NH4HCO3 and 80% acetonitrile (ACN). The ACN su-
pernatant and the NH4HCO3 fractions were combined and concentrated
in a SpeedVac concentrator (Bachofer, Reutlingen, Germany) (Table 2).
2.5. Mass spectrometry (MS) analysis
LC–MS/MS analyses were performed with a nano-liquid chromatog-
raphy system (Ettan MDLC; GE Healthcare, Munich, Germany) coupled
to a linear ion trap mass spectrometer (LTQ, Thermo Fisher Scientific,
MA, USA). Tryptic peptide solutions were reconstituted in 0.1% formic
acid, injected onto a C18 trap column (C18 PepMap100, 5 μm particle
size, 100 Å, 300 μm × 5 mm column size; LC Packings Dionex, Sunnyvale,
CA, USA) and subsequently separated by RP chromatography using an
analytical column (ReproSil-Pur C18 AQ, 3 μm; 150 mm × 75 μm,
Dr. Maisch, Ammerbuch-Entringen, Germany). Solvent A consisted of
Table 1
Age, gender and phenotype of animals included in this study.
Age
(month)
Gender Number of
animals
Genotype
wt BTC Wa5 BTC/Wa5
2–4 Female = 4;
male = 5
9 5 (f = 2;
m = 3)
4 (f = 2;
m = 2)
0 0
5–6 Female 17 3 7 3 4
8–12 Male 17 4 7 3 3
34 H. Schulz et al. / Experimental and Molecular Pathology 99 (2015) 33–38
3. 0.1% formic acid, and solvent B was composed of 84% acetonitrile in 0.1%
formic acid. Separation was performed using an 80-min gradient from
0% B to 30% B followed by a 30-min gradient from 30% B to 60% B. The
MS method consisted of a cycle combining one full MS scan with three
data dependent MS/MS events (35% collision energy). MS/MS data
were analyzed with Mascot version 2.4 (Matrix Science, Boston, MA)
using the following parameters: (i) enzyme, trypsin; (ii) fixed modifica-
tion, carbamidomethyl (Cys); (iii) variable modification, oxidation
(Met); (iv) peptide tolerance, 2 Da; (v) MS/MS tolerance, 0.8 Da;
(vi) peptide charge, 1+, 2+, and 3+; and (vii) instrument, ESI-TRAP.
As database the murine subset of the Swissprot (Release 2013_08)
was used. For validation of identifications and for spectral count quanti-
fication, the Scaffold software (V4.4.0, Proteome Software Inc., Portland,
Oregon, USA) was used.
2.6. Statistics
For growth and weight measurements, the mean value with stan-
dard error and independent two-tailed t-test was performed, with
p b 0.05 being considered significant. For multiple experiment compar-
ison, ANOVA table and Tukey's test were used with p b 0.05 being
regarded significant.
3. Results
We performed immunodetection in BTC (Fig. 1A–D), ERBB1/EGFR
(Fig. 1E–H), ERBB4 (Fig. 1I–L) and CD44v6 (Fig. 1M–P) of urinary blad-
der cross-sections derived from wild type (Fig. 1A, E, I, M), BTC trans-
genic (Fig. 1B, F, J, N), Wa5 (Fig. 1C, G, K, O) and BTC/Wa5 mice
(Fig. 1D, H, L, P). Strong immunostaining for BTC was detected in
microvessels of the stromal compartment and in umbrella cells of the
apical uroepithelial layer of the urinary bladder of BTC transgenic
(Fig. 1B) and BTC/Wa5 mice (Fig. 1D). In bladders collected from wild
type (wt; Fig. 1A) and Wa5 mice, (Fig. 1C), BTC was exclusively present
in umbrella cells of the uroepithelial lining but absent in the stromal
compartment. We studied the presence of the two known BTC receptors
ERBB1/EGFR and ERBB4. Bladder sections of all four genotypes
expressed immunoreactive ERBB1/EGFR in cells of the basal epithelial
layer of the bladder epithelium and in the microvascular endothelium
of the stromal bladder compartment (Fig. 1E–H). Of note, mice of the
Wa5 genotype expressed immunoreactive EGFR deficient in kinase ac-
tivity (Fig. 1E–H). Homogeneous immunostaining for ERBB4 was ob-
served in the basal and intermediate uroepithelial layer as well as in
cytosolic vesicles of umbrella cells (Fig. 1I–L). The cell adhesion mole-
cule CD44v6 was present throughout the bladder epithelium with
Fig. 1. Immunohistological detection of BTC (A–D), ERBB1/EGFR (E–H), ERBB4 (I–L) and CD44v6 (M–P) in urinary bladder sections of wild type (A, E, I, M), BTC transgenic (B, F, J, N), Wa5
(C, G, K, O) and BTC/Wa5 mice (D, H, L, P). Immunoreactive BTC was detected in and around microvessels of the bladder stroma and in umbrella cells of the urinary bladder of BTC trans-
genic (B) and BTC/Wa5 mice (D). In wild type (wt; A) and Wa5 mice (C), BTC was exclusively detected in umbrella cells but absent in the stroma. Immunoreactive ERBB1/EGFR was
present in the basal epithelial cell layer of the uroepithelium and in the microvascular endothelial cells of the stroma of all four genotypes studied (E–H). ERBB4 was homogeneously
expressed in the uroepithelium and in cytosolic vesicles of umbrella cells (I–L). Immunoreactive CD44v6 was present in the bladder epithelium with more intense immunostaining in
the basal cell layer (M–P). For negative controls, specific primary antibodies were replaced by a non-immune IgG of the respective species at the same concentration (Fig. 1A′–P′).
35H. Schulz et al. / Experimental and Molecular Pathology 99 (2015) 33–38
4. more intense immunostaining in the basal versus the apical
uroepithelial cell layer (Fig. 1M–P). Nuclear immunostaining for the
proliferation maker Ki67 was only observed occasionally in single cells
of the uroepithelium indicating a low percentage of actively proliferat-
ing cells in all bladder tissue compartments (data not shown).
Employing H&E stained (Fig. 2A) and ERBB4 immunostained bladder
tissue sections, we determined the height of the uroepithelium in 2–4,
5–6, and 8–12 months old mice of different genotypes. At 2–4 months
of age, we did not observe a difference in the height of the uroepithelial
layer between bladders from BTC and wild type mice (Fig. 2B). At
5–6 months of age, we detected a significant increase in the height
of the uroepithelium in BTC and in BTC/Wa5 mice versus wild type
and Wa5 mice (Fig. 2C). Similarly, at 8–12 months of age we observed
a similar significantly higher uroepithelium in BTC versus wild type
and Wa5 mice. The height of the uroepithelium of BTC/Wa5 mice was
also increased but failed to reach statistical significance (Fig. 2D).
We collected urine samples from male and female wild type and BTC
transgenic mice for LC–MS/MS analysis to determine the presence of
BTC. Mass spectrometric analysis did not reveal BTC in urine of either
male or female BTC transgenic mice. However, in wild type and BTC
transgenic mice we identified the presence of MUP1, 2, 3 and 6 in
urine samples collected from male mice at 2 months of age and female
mice at 8 months of age (Table 2). Coomassie stained gels showed a
dominant protein band between 15 and 20 kDa resembling major uri-
nary proteins (MUP) (Fig. 3). Male wild type and BTC transgenic mice
at 2 months (Fig. 3) showed a strong Coomassie stained protein band
of similar size. Similar levels of MUP1, 2, 3 and 6 levels were detected
in urine of wild type and BTC males (Table 2). By contrast, urine collect-
ed from female BTC mice at 8 months of age consistently showed a di-
minished Coomassie stained MUP protein band when compared to
urine from wild type females (Fig. 3). LC–MS/MS analysis confirmed
the sex-specific differences observed with the Coomassie protein stain
of urinary MUPs (Table 2).
4. Discussion
In the present study, we show the presence of BTC in the stromal
compartment and in umbrella cells of the uroepithelium of transgenic
mice with systemic over-expression of BTC (Schneider et al., 2005).
Strong BTC expression was detected in stromal microvascular vessels
which were prominent and frequently observed in the bladder stroma
of BTC transgenic and BTC/Wa5 mice but not wild type and Wa5 mice.
BTC has previously been associated with vascular remodeling by pro-
moting the growth and migration of vascular smooth muscle cells
Fig. 2. Representative image of a hematoxylin and eosin (H&E) stained mouse urinary bladder section with indicated measurements of uroepithelial height (red lines; A). Dot blots
showing the uroepithelial height distribution in cross-sectioned non-distended bladder tissues from wild type and BTC transgenic mice at 2–4 months of age (B) and wild type, BTC trans-
genic, Wa5 and BTC/Wa5 mice at 5–6 (D) and 8–12 months of age (E). BTC and BTC/Wa5 mice at 5–6 months of age displayed uroepithelial hyperplasia (D), as did BTC transgenic mice at
8–12 months versus wild type and Wa5 mice (E). BTC/Wa5 mice at 8–12 months of age reached borderline significance. Geometric symbols represent individual animals. *p b 0.05;
**p b 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Table 2
LC–MS/MS results with quantitative values (normalized spectral counts) indicating the
frequency of detection of specific peptides of MUP proteins in urine samples collected
from male and female BTC transgenic and wild type mice.
MUP WT, male BTC, male WT, female BTC, female
1 551.41 629.48 259.92 126.53
2 619.35 689.97 360.38 139.18
3 275.4 213.81 159.46 57.639
6 553.25 614.01 333.27 139.18
36 H. Schulz et al. / Experimental and Molecular Pathology 99 (2015) 33–38
5. (Mifune et al., 2004). BTC also increases the permeability of retinal
blood vessels in normo- and hyperglycemic mice in an EGFR-
dependent manner (Anand-Apte et al., 2010; Sugimoto et al., 2013).
This vascular effect of BTC may also contribute to the development of se-
vere pulmonary alveolar hemorrhages we observed in transgenic mice
over-expressing BTC (Schneider et al., 2005). The bladder epithelium
of BTC transgenic mice showed an expression pattern of the two BTC re-
ceptors ERBB1/EGFR and ERBB4 that was similar to wild type and Wa5
mice. In addition, the adhesion molecule CD44 variant 6 (CD44v6), an
epithelial marker down-regulated in transitional bladder cancer (Ross
et al., 1996; Iczkowski et al., 1998), was expressed similarly in the
uroepithelium of the different genotypes suggesting that over-
expression of BTC in the bladder did not alter the cell–cell connections
or predispose to bladder cancer. However, as early as 5 months of age
BTC transgenic mice of both sexes demonstrated significant hyperplasia
of the bladder epithelium. We had shown previously that BTC can in-
duce simple hyperplasia of mucosal surfaces in the intestine (Dahlhoff
et al., 2008) and the formation of BTC mediated hyperplastic gastric
polyps with the depletion of specific gastric cell types and a remodeling
of the gastric epithelium (Dahlhoff et al., 2012). The hyperplastic
uroepithelium retained its normal histological features. Importantly,
BTC/Wa5 mice carrying an antimorphic allele of the ERBB1/EGFR with
a non-functional mutation in the kinase domain (Lee et al., 2004)
showed a similar hyperplastic uroepithelium as well as an enriched
stromal microvasculature suggesting that, contrary to its role in bone
development and retinal vascular permeability (Schneider et al., 2009;
Mifune et al., 2004; Anand-Apte et al., 2010), the BTC-induced morpho-
logical changes in the urothelium and bladder stroma were not solely
dependent on the activation of ERBB1/EGFR but may also involve
ERBB4 which was consistently present in both compartments of the uri-
nary bladder (Dahlhoff et al., 2014).
The uroepithelium expresses EGF-like ligands (Mellon et al., 1996;
Freeman et al., 1997). We identified the stromal compartment and lu-
minal umbrella cells as source of BTC in the urinary bladder of BTC
and BTC/Wa5 mice. Despite the use of sensitive Western blot detection
and LC–MS/MS analysis, we were unable to identify BTC in the urine of
BTC and BTC/Wa5 mice. This suggested that the BTC detected in the
stromal compartment acted locally and was unable to penetrate the api-
cal umbrella cell layer which forms tight junctions that dynamically seal
and protect the underlying transitional epithelium and stroma from the
adverse effects of urine (Carattino et al., 2013). In addition, the amount
of BTC produced in umbrella cells may be too small for detection in
urine. It is conceivable that BTC serves local functions specific to umbrel-
la cells that require ERBB activation. Umbrella cells extend and contract
depending on the filling state of the urinary bladder and the associated
membrane plasticity requires EGFR activation (Balestreire and Apodaca,
2007). Furthermore, exocytosis by umbrella cells has recently been
shown to include ERBB1/EGFR transactivation and involves A1 adeno-
sine receptor triggered proteolytic cleavage of membrane-anchored
HBEGF by ADAM17 (Prakasam et al., 2014). An additional and unex-
pected finding of the mass spectrometric analysis of the urine samples
was the discovery that BTC affected the urinary levels of MUPs in a
sex-dependent manner. MUPs are produced in the liver and represent
a heterogeneous group of 18–20 kDa lipocalins. These proteins are ex-
creted into the urine where they reversibly bind different pheromone
compounds which are later released from scent marks (Beynon and
Hurst, 2004). Detected by the olfactory systems, MUP signatures are
an integral and complex communication tool among many animals, in-
cluding rodents, and trigger adaptive behavioral responses, regulate
physiological processes and affect nutrient metabolism (Zhou and Rui,
2010). While MUPs constitute the majority of proteins detectable in
mouse urine and 99% of all urinary proteins in male mice, there are
sex differences in the amount of MUPs detected in urine (Beynon and
Hurst, 2004; Hurst and Beynon, 2004). When compared to their wild
type female counterparts, female BTC transgenic mice contained signif-
icantly lower MUP levels in their urine. By contrast, male BTC and wild
type mice had similar amounts of urinary MUP levels at both 2 (Fig. 3)
and 6 months (data not shown) of age. To our knowledge, this is the
first report showing an association of an EGF-like ligand with dimin-
ished urinary MUPs. This provides an intriguing new rationale for a
role of BTC in modulating complex sex-specific behavioral and physio-
logical responses in rodents.
In conclusion, we provide evidence in BTC transgenic mice for a local
BTC over-expression in stromal and umbrella cells and demonstrate
that the resulting uroepithelial hyperplasia was not exclusively depen-
dent on the presence of ERBB1/EGFR. BTC was absent from urine of
BTC mice. However, BTC over-expression resulted in lower MUPs exclu-
sive in urine of female BTC mice, suggesting a possible novel role for BTC
in sex-specific odor-based communication among rodents.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
TK would like to thank the Cancer Research Society, Surgery Re-
search Fund and the Natural Science and Engineering Council of
Canada (NSERC) for financial support.
References
Anand-Apte, B., et al., 2010. Betacellulin induces increased retinal vascular permeability in
mice. PLoS One 5 (10), e13444.
Fig. 3. Representative Coomassie stained 12% SDS-PAGE gel of urine samples from four wild type and BTC transgenic male and female mice at 2 and 8 months of age, respectively. Urine
samples from male mice of both genotypes contained similar amounts of Coomassie stained major urinary protein which constitutes the majority of proteins in the urine of male mice
(Zhou and Rui, 2010; Hurst and Beynon, 2004) (A). As expected, urine derived from female mice had a lower content of MUPs (Hurst and Beynon, 2004). Female BTC transgenic mice
showed a further decrease in urinary MUPs (A). Western blot analysis for the detection of MUPs revealed specific bands at 18–21 kDa in female wild type but not BTC transgenic mice (B).
37H. Schulz et al. / Experimental and Molecular Pathology 99 (2015) 33–38
6. Balestreire, E.M., Apodaca, G., 2007. Apical epidermal growth factor receptor signaling:
regulation of stretch-dependent exocytosis in bladder umbrella cells. Mol. Biol. Cell
18 (4), 1312–1323.
Beynon, R.J., Hurst, J.L., 2004. Urinary proteins and the modulation of chemical scents in
mice and rats. Peptides 25 (9), 1553–1563.
Carattino, M.D., et al., 2013. Bladder filling and voiding affect umbrella cell tight junction
organization and function. Am. J. Physiol. Ren. Physiol. 305 (8), F1158–F1168.
Chow, N.H., et al., 1997. Expression patterns of erbB receptor family in normal urothelium
and transitional cell carcinoma. An immunohistochemical study. Virchows Arch. 430
(6), 461–466.
Dahlhoff, M., et al., 2008. Betacellulin stimulates growth of the mouse intestinal epitheli-
um and increases adenoma multiplicity in Apc+/Min mice. FEBS Lett. 582 (19),
2911–2915.
Dahlhoff, M., et al., 2012. A new mouse model for studying EGFR-dependent gastric
polyps. Biochim. Biophys. Acta 1822 (8), 1293–1299.
Dahlhoff, M., Wolf, E., Schneider, M.R., 2014. The ABC of BTC: structural properties and bi-
ological roles of betacellulin. Semin. Cell Dev. Biol. 28, 42–48.
el-Marjou, A., et al., 2000. Involvement of epidermal growth factor receptor in chemically
induced mouse bladder tumour progression. Carcinogenesis 21 (12), 2211–2218.
Freeman, M.R., et al., 1997. Heparin-binding EGF-like growth factor is an autocrine
growth factor for human urothelial cells and is synthesized by epithelial and smooth
muscle cells in the human bladder. J. Clin. Invest. 99 (5), 1028–1036.
Harris, R.C., Chung, E., Coffey, R.J., 2003. EGF receptor ligands. Exp. Cell Res. 284 (1), 2–13.
Hinkle, C.L., et al., 2004. Selective roles for tumor necrosis factor alpha-converting en-
zyme/ADAM17 in the shedding of the epidermal growth factor receptor ligand fam-
ily: the juxtamembrane stalk determines cleavage efficiency. J. Biol. Chem. 279 (23),
24179–24188.
Hurst, J.L., Beynon, R.J., 2004. Scent wars: the chemobiology of competitive signalling in
mice. Bioessays 26 (12), 1288–1298.
Iczkowski, K.A., Shanks, J.H., Bostwick, D.G., 1998. Loss of CD44 variant 6 expression differ-
entiates small cell carcinoma of urinary bladder from urothelial (transitional cell) car-
cinoma. Histopathology 32 (4), 322–327.
Jorissen, R.N., et al., 2003. Epidermal growth factor receptor: mechanisms of activation
and signalling. Exp. Cell Res. 284 (1), 31–53.
Kallincos, N.C., et al., 2000. Cloning of rat betacellulin and characterization of its expres-
sion in the gastrointestinal tract. Growth Factors 18 (3), 203–213.
Kim, J., Adam, R.M., Freeman, M.R., 2005. Trafficking of nuclear heparin-binding epider-
mal growth factor-like growth factor into an epidermal growth factor receptor-
dependent autocrine loop in response to oxidative stress. Cancer Res. 65 (18),
8242–8249.
Kramer, C., et al., 2007. Heparin-binding epidermal growth factor-like growth factor iso-
forms and epidermal growth factor receptor/ErbB1 expression in bladder cancer and
their relation to clinical outcome. Cancer 109 (10), 2016–2024.
Lee, D., et al., 2004. Wa5 is a novel ENU-induced antimorphic allele of the epidermal
growth factor receptor. Mamm. Genome 15 (7), 525–536.
Mellon, J.K., et al., 1996. Transforming growth factor alpha and epidermal growth factor
levels in bladder cancer and their relationship to epidermal growth factor receptor.
Br. J. Cancer 73 (5), 654–658.
Mifune, M., et al., 2004. Signal transduction of betacellulin in growth and migration of
vascular smooth muscle cells. Am. J. Physiol. Cell Physiol. 287 (3), C807–C813.
Naik, D.S., et al., 2011. Epidermal growth factor receptor expression in urinary bladder
cancer. Indian J. Urol. 27 (2), 208–214.
Prakasam, H.S., et al., 2014. A1 adenosine receptor-stimulated exocytosis in bladder um-
brella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor
transactivation. Mol. Biol. Cell 25 (23), 3798–3812.
Ross, J.S., et al., 1996. Expression of the CD44 cell adhesion molecule in urinary bladder
transitional cell carcinoma. Mod. Pathol. 9 (8), 854–860.
Rotterud, R., et al., 2005. Expression of the epidermal growth factor receptor family in
normal and malignant urothelium. BJ. Int. 95 (9), 1344–1350.
Sahin, U., et al., 2004. Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of
six EGFR ligands. J. Cell Biol. 164 (5), 769–779.
Sasada, R., et al., 1993. Cloning and expression of cDNA encoding human betacellulin, a
new member of the EGF family. Biochem. Biophys. Res. Commun. 190 (3),
1173–1179.
Schneider, M.R., 2014. The magnificent seven: epidermal growth factor receptor ligands.
Semin. Cell Dev. Biol. 28, 1.
Schneider, M.R., Wolf, E., 2009. The epidermal growth factor receptor ligands at a glance.
J. Cell. Physiol. 218 (3), 460–466.
Schneider, M.R., et al., 2005. Betacellulin overexpression in transgenic mice causes dispro-
portionate growth, pulmonary hemorrhage syndrome, and complex eye pathology.
Endocrinology 146 (12), 5237–5246.
Schneider, M.R., et al., 2008. Betacellulin regulates hair follicle development and hair cycle
induction and enhances angiogenesis in wounded skin. J. Investig. Dermatol. 128 (5),
1256–1265.
Schneider, M.R., et al., 2009. High cortical bone mass phenotype in betacellulin transgenic
mice is EGFR dependent. J. Bone Miner. Res. 24 (3), 455–467.
Seno, M., et al., 1996. Human betacellulin, a member of the EGF family dominantly
expressed in pancreas and small intestine, is fully active in a monomeric form.
Growth Factors 13 (3–4), 181–191.
Sugimoto, M., et al., 2013. Inhibition of EGF signaling protects the diabetic retina from
insulin-induced vascular leakage. Am. J. Pathol. 183 (3), 987–995.
Sunnarborg, S.W., et al., 2002. Tumor necrosis factor-alpha converting enzyme (TACE)
regulates epidermal growth factor receptor ligand availability. J. Biol. Chem. 277
(15), 12838–12845.
Waterman, H., Yarden, Y., 2001. Molecular mechanisms underlying endocytosis and
sorting of ErbB receptor tyrosine kinases. FEBS Lett. 490 (3), 142–152.
Yarden, Y., Sliwkowski, M.X., 2001. Untangling the ErbB signalling network. Nat. Rev. Mol.
Cell Biol. 2 (2), 127–137.
Zhou, Y., Rui, L., 2010. Major urinary protein regulation of chemical communication and
nutrient metabolism. Vitam. Horm. 83, 151–163.
38 H. Schulz et al. / Experimental and Molecular Pathology 99 (2015) 33–38
