GSK3 and PKB/Akt are associated with integrin-mediated regulation of PTHrP, IL-6
and IL-8 expression in FG pancreatic cancer cells
John J. Grzesiak1, Kathy C. Smith2, Douglas W. Burton2, Leonard J. Deftos2and Michael Bouvet1*
1Department of Surgery, University of California, San Diego and Veterans Affairs San Diego Healthcare System,
San Diego, CA, USA
2Department of Medicine (Endocrinology), University of California, San Diego and Veterans Affairs San Diego Healthcare System,
San Diego, CA, USA
We have demonstrated recently that PTHrP is upregulated in
pancreatic adenocarcinoma and that the ECM exerts regulatory
control, at least in part, over PTHrP expression. In our present
study, we examined the potential signaling interactions between
these 2 pathways. Our results demonstrate that, under serum-free
conditions, adhesion of FG pancreatic adenocarcinoma cells on Fn
is mediated by the ?5?1integrin, whereas adhesion to Type I
collagen is mediated by the ?2?1integrin. ?5?1integrin-mediated
adhesion to Fn results in a phenotype that includes a reduction in
cell proliferation, increased E-cadherin localization in cell–cell
contacts, increased ?-catenin localization throughout the cell, in-
hibition of haptokinetic cell migration, and increased expression of
PTHrP, IL-6 and IL-8 relative to ?2?1integrin-mediated adhesion
on Type I collagen. A phosphoprotein immunoblotting screen of
FG pancreatic cancer cells grown on either Fn or Type I collagen
indicates that GSK3 and PKB/Akt are differentially phosphory-
lated on these 2 substrates. These results implicate GSK3 and
PKB/Akt in the integrin-mediated regulation of PTHrP, IL-6 and
IL-8 in pancreatic cancer.
© 2004 Wiley-Liss, Inc.
Key words: ?-catenin; Lef/Tcf; fibronectin; Type I collagen; ECM
Pancreatic adenocarcinoma is a devastating disease character-
ized by the progressive accumulation of genetic mutations includ-
ing those of K-ras, CDKN2A, p53, BRCA2 and SMAD4/DPC4.1
Mutations of the SMAD4 gene in particular, result in the consti-
tutive activation of TGF? signaling and are thought to be respon-
sible for the extensive proliferation of stromal fibroblasts and
deposition of ECM (desmoplasia) that are hallmarks of this dis-
ease.1The ECM, interacting with cells through cell adhesion
receptors, in particular the integrins, has been shown to be a
critical regulator of important cell processes including angiogen-
esis, mitogenesis, migration and differentiation.2,3We have dem-
onstrated recently that specific ECM proteins, known to be aber-
rantly expressed in pancreatic adenocarcinoma, affect the
expression of the PTHrP axis.4
PTHrP is an onco-fetal protein expressed in normal tissues as
well as many malignancies, including pancreatic, breast, colon,
gastric, melanoma, lung and prostate cancers.5–11PTHrP seems to
play a role in cell growth, proliferation and angiogenesis,12–15
functioning primarily, but not exclusively, through a member of
the 7 membrane spanning and G protein-coupled cell surface
receptor family, the PTHrPR.16,17
Fn is an ECM protein found normally with a relatively even
distribution in the basement membrane of pancreatic ductal
cells.18–24In pancreatic cancer, Fn expression becomes down-
regulated in the basement membrane and sparsely expressed
throughout the ECM.19Our previous results demonstrated that
growth of FG pancreatic adenocarcinoma cells on Fn resulted in
a relative decrease in proliferation and increased expression of
PTHrP and the PTHrPR compared to Type I collagen after 96 hr
in serum-free culture.4Type I collagen, which is normally
expressed in the interstitium, is profoundly upregulated in pan-
creatic cancer.18–21,23,24The differential effects mediated by
these 2 ECM substrates were also characterized by morpholog-
ical changes. Whereas growth of FG cells on Fn substrates was
accompanied by “strong” cell–cell contacts, growth on Type I
collagen resulted in “loose” cell–cell contacts.4
We extend our initial observations by examining the potential
molecular mechanisms responsible for the regulation of PTHrP
expression on these 2 important ECM components in pancreatic
cancer. Our results indicate that GSK3 and PKB/Akt intracellular
signaling is associated with the integrin-mediated regulation of
PTHrP, IL-6 and IL-8 in FG pancreatic cancer cells.
Material and methods
FG cells are a fast-growing (FG), metastatic variant of the
pancreatic adenocarcinoma cell line, COLO-357. FG cells were
used for these studies because their integrin profile is known,25,26
they express relatively high levels of PTHrP (unpublished obser-
vations) and they were used in our previous studies.4Cells were
cultured in DMEM supplemented with 10% FBS in a humidified
atmosphere containing 5% CO2at 37°C.
Cell culture assays
FG cells were serum-starved 24 hr before assay in DMEM
supplemented with 1 mg/ml BSA (Sigma, St. Louis, MO). Six-
well culture plates (Becton Dickinson, Franklin Lakes, NJ), not
treated for tissue culture, were coated with either Fn (1–48 ?g/ml)
or Type I collagen (3–25 ?g/ml) in PBS for 24 hr at RT (Chemi-
con, Temecula, CA). These ECM coating concentrations have
been shown previously to promote adhesion or migration of FG
cells.4,25,26ECM-coated wells were then washed twice with PBS
and blocked with 1 mg/ml BSA in PBS for 1 hr at 37°C. Cells were
trypsinized, treated with 1 mg/ml soybean trypsin inhibitor
(Sigma), followed by 3 PBS washes. Cells were resuspended in
serum-free medium and seeded at 2.5 ? 105/ml, 2 ml/well. Cul-
tures were then incubated and harvested at the indicated time
Inhibition of cell attachment assays
Inhibition of attachment assays were carried out as described
previously.27Briefly, 96-well microtiter plates were previously
Abbreviations: AES, amino terminal enhancer of split; Bit1, Bcl-2
inhibitor of transcription; BSA, bovine serum albumin; BRCA2, breast
cancer 2; CDKN2A, cyclin-dependent kinase inhibitor 2A; CDK1, cyclin-
dependent kinase 1; DMEM, Dulbecco’s modified Eagles’ medium; ECM,
extracellular matrix; ELISA, enzyme-linked immunosorbent assay; FBS,
fetal bovine serum; Fn, fibronectin; GSK3, glycogen synthase kinase 3; IL,
interleukin; ILK, integrin-linked kinase; Lef/Tcf, lymphoid enhancer fac-
tor/T-cell factor; mAb, monoclonal antibody; PKB?, protein kinase B?;
PTHrP, parathyroid hormone-related protein; PTHrPR, parathyroid hor-
mone-related protein receptor; Rb, retinoblastoma; RT, room temperature;
SMAD4/DPC4, mother’s against decapentaplegic, deleted in pancreatic
carcinoma, locus 4; TGF?1, transforming growth factor ?1.
Grant sponsor: Department of Veterans Affairs; Grant sponsor: National
Institute of Health; Grant number: DK60588, AR47347.
*Correspondence to: Department of Surgery (112-E), University of
California, Veterans Affairs San Diego Healthcare System, 3350 La Jolla
Village Drive, San Diego, CA 92161. Fax: ?858-552-4352.
Received 18 June 2004; Accepted after revision 15 September 2004
Published online 17 December 2004 in Wiley InterScience (www.
Int. J. Cancer: 114, 522–530 (2005)
© 2004 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
coated with Type I collagen (10 ?g/ml) or Fn (25 ?g/ml) and
unbound sites blocked with 1 mg/ml BSA as described above. FG
cells (5 ? 104) for Type I collagen or 105FG cells for Fn were
added to each well in serum-free DMEM supplemented with 1
mg/ml BSA. Purified monoclonal antibodies were added at a final
concentration of 25 ?g/ml in the serum-free medium described
above. After 1 hr incubation at 37°C, media were removed. At-
tached cells were fixed with 4% paraformaldehyde in PBS and
stained with 0.5% toluidine blue in 3.7% formaldehyde. Stained
cells were subsequently solubilized with 20% SDS and the absor-
bance read at 595 nm.
Immunofluorescence studies were conducted as described pre-
viously.28Briefly, 13 mm glass coverslips were coated with Type
I collagen (10 ?g/ml) or Fn (25 ?g/ml) overnight at RT, and
subsequently blocked with 1 mg/ml BSA in PBS for 1 hr at 37°C.
Serum-starved FG cells were plated on coated coverslips in serum-
free DMEM supplemented with 1 mg/ml BSA at a density of 2.5 ?
105cells/well for 24 hr at 37°C. The cells were then fixed with 4%
paraformaldehyde in PBS for 5 min at RT and permeabilized with
0.1% Triton X-100 in PBS for 10 min at RT. After incubation with
1% normal goat serum/1% BSA in PBS for 10 min at RT, the
coverslips were incubated with anti-E-cadherin or anti-?-catenin
mAbs (Chemicon) at 10 ?g/ml in 1% normal goat serum/1% BSA
in PBS for 30 min at RT. After rinsing with PBS, coverslips were
incubated with fluorescein-isothiocyanate-conjugated secondary
antibody (goat anti-mouse IgG at 1:100 in 1% normal goat se-
rum/1% BSA in PBS) (Jackson ImmunoResearch Laboratories,
Inc., West Grove, PA) for 10 min at RT. Coverslips were rinsed,
mounted and fluorescence microscopy was carried out with an
Olympus BX-60 microscope equipped with a Spot Digital Imaging
package (Diagnostic Instruments, Inc., Burlingame, CA).
Migration assays were conducted using the modified Boyden
chamber as described previously.27Briefly, the chamber consists
of 2 compartments separated by a filter and migration was mea-
sured by counting the number of cells crossing the membrane
through pores of defined size. Lower chambers were filled with
serum-free DMEM (Invitrogen, Carlsbad, CA) supplemented with
1 mg/ml BSA pore polycarbonate membrane filters (Neuro Probe,
Inc., Gaithersburg, MD), that were coated with either Type I
collagen (10–25 ?g/ml) or Fn (3–25 ?g/ml), were then placed on
top of the lower chambers, and the upper chambers were secured
in place. Upper chambers were filled with 5 ? 104FG cells that
were serum-starved 24 hr before assay, in the same media de-
scribed above. Lower chamber final volumes were 27 ?l and the
upper chambers were 50 ?l. The entire apparatus was then incu-
bated for 17–22 hr at 37°C. After the incubation period, the filters
were fixed in methanol and stained with 0.5% toluidine blue in
3.7% formaldehyde. Excess stain was washed away with water, the
attached cells on the upper side of the filters were mechanically
removed using wet, cotton-tipped applicators. The migratory cells
on the underside of the filters were quantitated by counting 4 high-
powered fields (100? magnification) per well using an inverted light
microscope (Olympus BH 2). Photomicrographs were imaged using a
Nikon Eclipse TE 300 inverted light microscope (Nikon Inc.,
Melville, NY) equipped with a Spot Digital Imaging package.
At the indicated time points, media were harvested and cell
extracts were prepared by sonication in lysis buffer containing 0.25
M Tris, pH 7.4, 0.25% Nonidet P-40 and 2 mM EDTA. The
insoluble fractions were pelleted by centrifugation at 16,000g for
15 min and the soluble fractions were transferred to a fresh tube.
Total cell protein was then measured using a modified Bradford
protein assay with BSA as the standard (Bio-Rad Laboratories,
Inc., Hercules, CA). PTHrP was measured with an in-house radio-
immunoassay, using modifications described previously.29Human
PTHrP 1-34 peptide (Bachem, King of Prussia, PA) was used as
the assay standard. Rabbit antisera raised against the peptide was
used in a non-equilibrium immunoassay format. PTHrP 1-86
(Bachem) was used to prepare tracer by chloramine-T-radioiodi-
nation. Lack of cross-reactivity in the assay for at least a 100-fold
excess of peptide was demonstrated for non-corresponding PTHrP
peptides, calcitonin, calcitonin gene-related peptide and human
and rat atrial natriuretic peptide and bone natriuretic peptide. All
samples were assayed in multiple dilutions that paralleled the
corresponding PTHrP standard. The intra- and interassay varia-
tions were 7 and 12%, respectively.30
To assess IL-6 and IL-8 levels, media from the cell culture
experiments described above were assayed using specific sand-
wich ELISA according to manufacturer’s recommendations as
described previously (BioSource International, Camarillo, CA).31
Briefly, 96-well microtiter plates (Immulon 4 HB) were coated
with anti-human IL-6 or IL-8 mouse monoclonal antibodies (0.6
?g/ml) and incubated overnight at 4°C. The captured plates were
blocked for 2 hr and washed 4? with PBS containing 0.1% Tween
20. Serial dilutions of recombinant IL-6 and IL-8 and test samples
were added to the wells followed immediately by the addition of
50 ?l/well of the biotinylated anti-human IL-6 or IL-8 detection
monoclonal antibody (0.4 ?g/ml). After subsequent incubation
with streptavidin ?-galactosidase conjugate (Calbiochem), the flu-
orescent substrate for ?-galactosidase, methyl umbelliferone ga-
lactopyranoside (MUG), was added. The fluorescence signal gen-
erated by the final product, methyl umbelliferone (4 MU), was
detected by reading microtiter plates with filters for 355 nm
FIGURE 1 – FG pancreatic cancer cell adhesion
on Fn and Type I collagen is mediated by the
?5?1and ?2?1integrins, respectively. Inhibition
of FG cell attachment was conducted on Fn (25
?g/ml) or Type I collagen (10 ?g/ml) coated
welts with purified monoclonal antibodies di-
rected against the indicated specific integrin sub-
units at a final concentration of 25 ?g/ml (see
Material and Methods). Black bars ? Type I
collagen; grey bars ? Fn. Data are expressed as
the percent maximum for each substrate and rep-
resent the mean ? SEM of 3 experiments carried
out in triplicate. *p ? 0.05.
ECM REGULATION OF PTHrP, IL-6 AND IL-8 IN FG CELLS
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