J Neurooncol (2011) 103:277285
DOI 10.1007/s11060-010-0399-y
LABORATORY INVESTIGATION - HUMAN/ANIMAL TISSUE
Analysis of transforming growth factor b receptor expression
and signaling in higher grade meningiomas
Mahlon D. Johnson Aubie K. Shaw
Mary J. OConnell Fraser J. Sim
Harold L. Moses
Received: 23 June 2010 / Accepted: 31 August 2010 / Published online: 19 September 2010
Springer Science+Business Media, LLC. 2010
Abstract TGF-b receptors (TGF-bRs) inhibit growth of
many cell types. Loss of TGF-bRs or its signaling com-
ponents have been found in several human malignancies.
The expression and the role of TGF-bRs in regulating
anaplastic meningioma growth has not been studied. Real
time PCR found TGF-b RIII expression significantly lower
in five grade III compared to eight grade I and eight
grade II tumors (P = 0.0481). By western blot analysis,
TGF-bRI was detected in the four fetal and adult lepto-
meninges, all 18 grade I, 14 grade II and six grade III
meningiomas. TGF-bRII was detected in none of the lep-
tomeninges, 55% of grade I, 71% of grade II and weak to
negative in five of six the grade III meningiomas analyzed.
TGF-bRIII immunoreactivity was not detected in the fetal
meninges but was detected in 94% of grade I, 70% of grade
II and 67% grade III tumors. Phospho-SMAD 3 and Smad
7 were detected in nearly all tumors. TGF-b1 had no effect
on PDGF-BB stimulation of DNA synthesis in six of seven
WHO grade II and the grade III cells. It produced an
increase in phosphorylation of SMAD 3 and p38MAPK in
two of four and p44/42MAPK in three of four grade II cells
showing no change in DNA synthesis after treatment. Thus,
M. D. Johnson (&)
M. J. OConnell
Department of Pathology, Division of Neuropathology,
University of Rochester Medical Center, 601 Elmwood Ave.
Box 626, Rochester, NY 14642, USA
e-mail: mahlon_johnson@urmc.rochester.edu
F. J. Sim
Center for Translational Neuromedicine, Department of
Neurology, University of Rochester School of Medicine,
University of Rochester Medical Center, Rochester, NY, USA
A. K. Shaw
H. L. Moses
Department of Cancer Biology, Vanderbilt Ingram Cancer
Center, Vanderbilt University, Nashville, TN, USA
only attenuated TGF-bRIII expression and TGFB growth
inhibition may occur in select higher grade meningiomas.
Nonetheless, restoring TGF-b inhibition of meningioma
cell proliferation may be an important objective in the
design of new chemotherapies for these tumors.
Keywords Meningioma
Anaplastic meningioma

TGF-b receptors
TGF-b
SMAD 3
MAPK
Introduction
The transforming growth factor-bs (TGF-b) represents an
important family of peptides that inhibit proliferation of
nearly all normal epithelial cells, mesenchymal cells and
cells from some low grade neoplasias [1, 2]. However, loss
of TGF-b inhibitory effects is seen in higher grade tumors
[24]. In many cases, this is due to loss or inactivation of
TGF-b type I receptor (TGF-bRI), TGF-b type II receptor
(TGF-bRII) or signaling components such as SMAD 2, 3 and
4 [24]. Atypical and anaplastic meningiomas have not been
evaluated for defects in TGF-bR expression or signaling.
The TGF-b superfamily includes three closely related
25 kDa dimeric, disulfide bonded ligands [1, 5]. TGF-b
effects are largely transduced through binding to the
TGF-b type II receptor (TGF-bRII) that then complexes
with the TGF-b type I receptor phosphorylating and
activating its serine/threonine kinase (TGF-bRI) [6].
TGF-bRIII i.e. betaglycan, is a membrane glycoprotein
which facilitates TGF-b binding to the TGF-bRII [2]. The
activated TGF-bRI receptor phosphorylates a number of
membrane/cytoplasmic proteins which transduce growth
regulatory signals to the nucleus [6]. Previously, we have
shown that TGF-b1, 2 and 3 as well as TGF-b receptors I,
II and III are expressed in human leptomeninges and World
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Health Organization (WHO) grade I meningiomas [79].
WHO grade I meningioma cells synthesize and release all
three (presumably latent) TGF-b isoforms [7]. TGF-b is
also present in human CSF that bathes meningiomas [8, 10,
11] and in media conditioned by leptomeningeal cells [8].
In vitro, TGF-b1 reduced basal proliferation in 47% of
meningioma cell cultures from 15 WHO grade I meningi-
omas [7, 9] and attenuated epidermal growth factor induced
proliferation in five of six [9]. Thus, paracrine or autocrine
release of TGF-b1 from leptomeninges or meningioma
cells may tonically suppress growth of human leptome-
ningeal and WHO grade I meningioma cells by activation
of these receptors. TGF-b1 in cerebrospinal fluid also may
limit grade I meningioma growth [9]. This raises the pos-
sibility that loss of TGF-b inhibitory effects may contribute
to progression of meningioma cells.
The main signal effectors for TGF-b are the SMADs 2 and
3 [12, 13]. SMAD 3 is activated by TGF-b and regulates cell
proliferation [2, 5]. Activation of the TGF-bR II phosphor-
ylates the type I receptor cytoplasmic domain which, in turn,
phosphorylates the carboxyterminal domain of SMAD 2 and
SMAD 3 resulting in their heteromerization with SMAD 4 in
the cytoplasm. The activated SMAD 2, 3 and 4 complex
translocates to the nucleus where they interact with and sta-
bilize transcriptional complexes of numerous transcription
factors [3, 12, 13]. Loss of SMAD 2, 3 and 4 has been asso-
ciated with several forms of cancer [1, 2]. In WHO grade I
meningioma cells TGF-b signaling is associated with TGF-b1
phosphorylation of SMAD 2/3 [9]. Nonetheless, the role of
SMAD 2, 3 and 4 in transduction of growth regulatory signals
in WHO grade II and III meningiomas is unknown.
Under some circumstances, TGF-b utilizes other sig-
naling pathways including the RAF- MAPK/Erk kinase-
1(MEK-1)-mitogen-activated protein kinase (MAPK)
pathways [1420] and phosphoinositide 3 kinase (PI3K)-
protein kinase B/Akt-p70S6K [2124] and p38-JNK
pathways [18, 23]. While these pathways do not appear
activated by TGF-b1 in WHO grade I meningiomas, they
have been found to be activated by some cytokines in grade
II and III meningiomas [25, 26]. Nonetheless, their role in
transducing TGF-b signals in higher grade meningiomas
has not been studied.
In the present study, we evaluated whether grade II or III
meningiomas show a loss of TGF-bRs or alterations in
known TGF-b signaling pathways.
Materials and methods
Meningioma tissue
Frozen normal leptomeninges from four human fetuses and
one adult and tissue from 37 sporadic meningiomas
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J Neurooncol (2011) 103:277285
including 18 grade I, 14 grade II and six grade III menin-
giomas [27] were obtained primarily from the Cooperative
Human Tissue Network (Philadelphia, PA) or collected at
URMC with Institutional Review Board approval. Patient
and tumor characteristics are listed in Table 1.
RNA analysis of TGF-b RI, TGF-b RII and TGF-b
RIII, SMAD 7, PAI-1 and HGF in meningioma tumors
RNA was isolated from eight WHO grade I, eight WHO
grade II and five WHO grade III meningiomas using Trizol
reagent (Invitrogen) followed by DNAse (Promega) fol-
lowing manufacturer specifications. One microgram of
total RNA was reverse transcribed to generate cDNA using
M-MLV reverse transcriptase (Invitrogen). Relative
mRNA quantity was determined by real-time PCR using
Sybr green quantitation with iCycler instrumentation and
software (BioRad, Hercules, CA). Plasminogen activator
inhibitor type 1 gene (PAI-1) and hepatocyte growth factor
(HGF) are two genes activated by TGF-bI [28, 29]. Primer
sequences are available by contacting the authors. Gene
expression was assessed relative to GAPDH gene expres-
sion and analyzed by KruskalWallis non-parametric test-
ing and unpaired t-tests with the software program
GraphPad Prizm v 5.0c.
Western blots
Protein from meningiomas was obtained by mechanical
homogenization in RIPA Lysis Buffer (Upstate Biotech-
nology) with 1:100 Protease Inhibitor Cocktail (Sigma)
then frozen at -85C. The tissue slurries were thawed on
ice and vortexed vigorously. Solids were removed by
centrifugation. Protein concentrations were quantified
using a Bradford assay (BioRad Protein Assay reagent),
then 35 lg protein from each was loaded on 7.5% acryl-
amide gel then transferred to 0.45 lm nitrocellulose
membrane. The membrane was blocked overnight at 4C in
5% milk in TrisCl buffer with Tween 20 then incubated
with monoclonal antibodies to: TGF-b RI, TGF-b RII,
TGF-b RIII, p- SMAD 3, SMAD 7 (all R and D, Minne-
apolis) and Merlin (Cell Signaling Technology, Beverly,
MA).
Meningioma cell cultures
Primary meningioma cell cultures were established from
seven grade II and one grade III meningiomas as described
previously (Table 2) [30]. For experiments, only early
passages i.e. passage 25 were used. For immunocyto-
chemical characterization, meningioma cell cultures were
plated onto 4-well microscope slides (Nalgene NUNC Int.,
Rochester, NY) for 1 day in supplemented DMEM then
J Neurooncol (2011) 103:277285 279

Table 1 Leptomeninges and meningiomas for TGF-beta receptor fixed in formalin. Immunocytochemistry was performed
Western blots and/or PCR using a mouse monoclonal antibody against epithelial
Leptomeninges/ Age/ Location Classification Merlin membrane antigen (EMA) (prediluted, DAKO, Carpente-
meningioma gender (Western ria, CA) and in most cases Desmoplakin (1:200, Abcam
blots) Inc. Cambridge Mass) using the avidinbiotin-horseradish
L1 16 week Convexity Normal n.d. peroxidase method. The presence or loss of Neurofibro-
L2 20 week Convexity Normal n.d. matosis-2 gene product Merlin was assessed by western
L3 22 week Convexity Normal ? blot. (Table 3).
L4 23 week Convexity Normal n.d.
L5 Adult Convexity Normal n.d. TGF-b1 effects on meningioma cell proliferation
1 48 F R Sphenoid Meningo I ? and signaling
2 53 F R Convexity Mixed I ?
3 51 F Convexity Meningo I ? Primary cultures from seven grade II and one grade III
4 66 F R Frontal Trans I meningiomas were also plated in 96-well plates, serum
5 71 F R Convexity Meningo I ? deprived overnight then treated with DMEM, or DMEM
6 57 F L Frontal Meningo I with TGF-b1 (10 ng/ml), PDGF-BB (10 ng/ml) or
7 42 M L Frontal Trans I ? PDGF-BB (10 ng/ml) and TGF-b1 (10 ng/ml) for 72 h.
8 46 F Convexity Meningo I ? Cells from four WHO grade II meningiomas were also
9 F R Frontal Fibro I ? treated as above for 24 h. Cells from MC3 and 4 were also
10 56 F Convexity Trans I ? treated with PDGF and 1.0, 5.0 or 10 ng/ml TGF-b1. Cells
11 35 F R Temporal Trans I ? were subsequently analyzed in quadruplicate by CyQUANT
12 70 F Convexity Mixed I Cell Proliferation assay (Invitrogen, Carlsbad, CA).
13 40 F R Parietal Fibro I CyQUANT fluorescence dye binds nucleic acid. Two hun-
14 71 F R frontal Meningo I ?
dred microliters of CyQUANT GR solution was added to
15 50 M Sphenoid Fibro I ?
each well for 5 min at room temperature according to assay
specifications. Fluorescence, measured at an excitation
16 79 F L Sphenoid Meningo I
wavelength of 480 and emission wavelength of 520 nm was
17 38 F R Temporal Meningo I
measured by a plate reader. Differences between treatment
18 67 F Post fossa Trans I
groups were analyzed by unpaired, two-tailed t-tests (In Stat,
19 85 M L Temporal Trans II ?
Sigma, St. Louis).
20 67 M Frontal Trans II ?
To evaluate TGF-b1 signaling, cells from four WHO
21 73 M Convexity Menino/trans II ?
grade II meningiomas were also treated as above for 4 h
22 97 F Convexity Meningo II ?
then evaluated by Western blot for phosphorylation/acti-
23 14 M Frontal Fibrous II ?
vation of Smad 3, p-38MAPK and p44/42.
24 51 F Rt. Frontal Trans II ?
25 35 F L frontal Fibrous II
26 43 M T4 Trans II ?
Results
27 39 F R frontal Microcystic II ?
28 49 F Sella Meningo II ?
RNA analysis of TGF-b RI, TGF-b RII and TGF-b
29 54 F L1 Spine Fibrous II ?
RIII, SMAD 7, PAI and HGF in meningioma tumors
30 91 F L Frontal Meningo II ?
31 60 F Parasaggital Mixed II
Real-time PCR detected RNA for TGF-b RI, TGF-b RII
32 70 F L Frontal Trans II ?
and TGF-b RIII in eight of eight WHO grade I and eight of
33 26 F Skull base Ana III ?
eight WHO grade II and five of five WHO grade III
34 79 M L Frontal Trans III ?
meningiomas. TGF-b RI was not significantly lower in the
35 70 F Parietal Fibrous III
three grades (P = 0.2573) and was not statistically different
36. 69 M R Frontal Ana III
between grade I and III by t-test (P = 0.2425). TGF-b RII
37. 72 F R temporal Ana III n.a. was not significantly lower in the three grades
38. 74 M R Frontal Ana III ? (P = 0.8728) and was not statistically different between
n.a. not analyzed; n.d. not done grade I and III by t-test (P = 0.4255). TGF-b RIII was

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Table 2 Sources of
Leptomeninges/ Age/gender Location Classification WHO grade
meningioma cell cultures
meningioma

MC1 79 F Frontal Meningothelial II

MC2 (M26) 51 F R frontal Transitional II
MC3 (M27) 35 F L frontal Fibrous II
MC4 (M28) 43 F T4 Ttransitional II
MC5 (M29) 43 F R Frontal Microcystic II
MC6 74 M Ethmoid Secretory II
MC7 57 F L frontal Transitional II
MC8 11 F Convexity Papillary III

Table 3 Summary of TGF-bR protein, RNA and response to TGF-bI in meningiomas

Grade I meningioma protein Grade II Grade III mRNA differences in
meningioma meningioma grade I and II vs grade III
protein protein meningiomas

TGF-bRI 18/18 14/14 6/6 P = 0.257

TGF-bRII 10/18 11/14 5/6 P = 0.872
TGF-bRIII 17/18 10/14 4/6 P = 0.048
p-SMAD3 18/18 13/14 5/6 n.a.
SMAD7 18/18 14/14 5/6 P = 0.143
Response to TGF-bI Reduced basal proliferation in 7 of 15 (7); No growth No growth
attanuated stimulated proliferation inhibition inhibition
in 5 of 6 (9)
n.a. not analyzed

significantly different (P = 0.0481) and approached sig-
nificance between grade I and III by t-test (P = 0.1971)
(Fig. 1). PAI-1 expression was not statistically different
between grade I and III. HGF was also similar between
groups (P = 0.2724) and between grade I and III by t-tests
(P = 0.3173). SMAD 7 expression trended lower but was
not significant in the three grades (P = 0.1432) and
between grade I and III by t-test (P = 0.2406).
SMAD 7 was detected in all grade I, 13 of 14 grade II and 5
of 6 grade III tumors (Fig. 2).
Merlin was detected in 11 of 18 (61%) of grade I, 12 of 14
(86%) and 3 of 5 (60%) meningiomas (Table 1), primarily in
meningothelial but showed no correlation with the presence
of TGF-bRI, TGF-bRII or TGF-bRIII protein (Table 1).
Primary meningioma cell cultures

Western blot analysis of TGF-b RI, TGF-b RII, TGF-b
RIII, p-SMAD 3, SMAD 7 and Merlin
By Western blot analysis, TGF-bRI was detected in the 16,
20, 22, 23 week fetal and one adult leptomeningeal sample
(not shown), all 18 of 18 WHO grade I meningiomas, 14 of
14 grade II and 6 of 6 grade III meningiomas analyzed.
TGF-bRII was detected in none of the leptomeningeal
samples (not shown), 10 of 18 WHO grade I meningiomas,
11 of 14 grade II and weak to negative at 75 kDa in the 5 of
6 grade III meningiomas analyzed. TGF-bRIII was not
detected in the 16, 20, 22, 23 week fetal leptomeninges but
was detected in 17 of 18 grade I and 10 of 14 grade II
(variable to weak in four) and four of six grade III tumors
(Fig. 2).
Phospho-SMAD 3 was detected in all 18 of 18 grade I,
13 of 14 grade II and 5 of 6 WHO grade III meningiomas.
The meningioma cell cultures appeared homogeneous and
were populated by large polygonal cells with irregular
borders and central round to oval nuclei and nucleoli. By
immunocytochemistry, meningioma cells exhibited exten-
sive EMA and variable desmoplakin immunostaining in
nonconfluent dispersed cells of each meningioma cell
culture. By western blot, Merlin was detected in the lep-
tomeningeal and five of seven meningioma cell cultures
(MC2, MC5, MC6, MC8 and MC11) evaluated but not in
MC4 or MC7.
TGF-b1 effects on meningioma primary cell culture
proliferation and signaling
At 24 h TGF-b1 (10 ng/ml) had no effect on basal cell
proliferation, based on DNA analysis with CyQUANT, in
four of four WHO grade II meningioma cells. TGF-b1 had

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Fig. 1 RNA analysis of TGF-b

RI, TGF-b RII and TGF-b RIII,
SMAD 7, PAI and HGF in
WHO grade I, II and III
meningiomas. Messenger RNA
concentrations 8 WHO grade I,
8 WHO grade II and 5 WHO
grade III meningiomas relative
to GAPDH were analyzed by
KruskalWallis test (mean and
standard error). TGF-b RIII
mRNA was reduced in WHO
grade III meningiomas
compared to grade I and II
tumors. Plasminogen activator
inhibitor type 1 gene (PAI-1)
and hepatocyte growth factor
(HGF) are two genes activated
by TGF-bI

no effect on PDGF-BB-mediated stimulation of prolifera- Discussion

tion in the four meningiomas.
At 72 h, TGF-b1 (10 ng/ml) had no effect on basal cell
proliferation, based on DNA analysis with CyQUANT, in
seven of seven WHO grade II and the WHO grade III
meningioma cells. TGF-b1 significantly reduced PDGF-BB
stimulation of DNA synthesis in the 22 week fetal lepto-
meninges but had no effect on PDGF-BB stimulation in six
of seven WHO grade II and the WHO grade III meningioma
(Fig. 3, and not shown).
By Western blot, TGF-b1 (10 ng/ml) treatment pro-
duced an increase in phosphorylation of SMAD 3 in two of
four WHO grade II meningioma cultures which had shown
no change in DNA synthesis after treatment. Compared to
controls, TGF-b1 alone or with PDGF-BB also increased
phosphorylation of p38MAPK in two of four and p44/42
MAPK in three of four WHO grade II meningioma cell
cultures (Fig. 4).
Findings from the present study suggest that higher grade
meningiomas do not exhibit reduced expression of
TGF-bRI but may, in a limited number of tumor, have
altered TGF-bRIII expression and show attenuated TGF-b1
growth inhibition. TGF-bRIII mRNA, and in some cases
TGF-bRIII protein was significantly reduced in WHO
grade III compared to lower grade meningiomas. Because
TGF-b effects are initiated by binding to the TGF-b type II
and III receptors, a genetic or epigenetic reduction in the
concentration of receptors might result in attenuated
TGF-b growth inhibition permitting less restrained growth
of higher grade meningiomas. Loss of TGF-bRs has also
been described in numerous other malignancies. Missense
or frameshift mutations in the TGF-bRI has been demon-
strated in head and neck, esophageal, breast and ovarian
carcinomas [2, 3, 3135]. Epigenetic decreased expression

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Fig. 2 Western blot analysis TGF-b RI, TGF-b RII, TGF-b RIII,
p-SMAD 3 and Smad 7 in meningiomas. Frozen tissue from WHO
grade I (a, 19), grade II (b, 2432) and grade III (c, 3338)
meningiomas were analyzed. TGF-b RII are expressed as 71 and
in TGF-bRI has been found in gastric carcinoma [2].
Inactivating mutations in TGF-bRII, often in tumors with
microsatellite instability, are far more common, being
found in head and neck carcinomas, esophageal, pulmon-
ary, biliary, gastric, colonic and ovarian carcinomas [2, 3,
33, 3639]. Loss or downregulation of the TGF-bRIII may
be the most prevalent receptor changes in malignancies
having been identified in neuroblastomas, ovarian, endo-
metrial and prostate carcinoma, renal and non-small cell
pulmonary cancers [4047]. Loss of TGF-bRIIIs may also
permit progression of breast cancer [48]. In addition,
recent, comparative genomic hybridization identified a
2.3-fold reduction in TGF-bRIII gene copy in high pro-
liferative meningiomas [49].
TGF-b1 had no detectable effect on cell proliferation in
six of seven WHO grade II and the grade III meningioma.
In contrast, we have previously found that TGF-b1 pro-
duced mild growth inhibition in some WHO grade I [7, 9].
Although it was only possible to culture one WHO grade
III meningioma over a number of years, collectively the
findings raise the possibility that reduced TGF-b1 growth
inhibitory signaling is present in at least some higher grade
tumors. Additional larger studies, may clarify whether a
loss of TGF-b signaling contributes to anaplasia in
meningiomas.
TGF-b1 stimulated an increase in p38 phosphorylation
in two and p44/42 MAPK in three meningioma cell cul-
tures from grade II meningiomas. This raises the possibility
that p38 and/or p44/42 MAPK transduce some TGF-b
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J Neurooncol (2011) 103:277285
75 KDa bands. TGF-b RIII is expressed as multiple 100 to 200 kDa
bands. TGF-bRII was detected in 55% of grade I, 71% of grade II and
was weak to negative in the grade III meningiomas analyzed
effects in meningioma cells with low, but still partially
functional receptors. Recently, the extent of SMAD and/or
p44/42 MAPK activation by TGF-b1 have been correlated
with TGF-b RII levels with lower levels favoring activa-
tion of the latter pathway [50]. This raised the possibility
that as meningiomas progress and TGF-b RII levels
decline, non-SMAD, non-inhibitory pathways regulating
other cell functions may occur.
SMAD 7 is a known inhibitor of TGF-b signaling and
part of complex feedback loops regulating TGF-b effects
[2, 3]. Overexpression of SMAD 7, compromising TGF-b
effects, has been found in thyroid and endometrial carci-
nomas [5153]. Nonetheless, in the present study, SMAD
7 expression was not increased or significantly different
between grades of meningiomas and SMAD 7 protein was
detected in all of the WHO grade I, II and III tumors. In
contrast, a recent comparative genomic hybridization
study found a three-fold reduction in SMAD 7 gene
expression in high grade compared to low grade menin-
giomas [40]. Future studies may clarify the role, if any, of
SMAD7 in modulating TGF-b regulation of meningioma
growth.
Expression of TGF-bRs, SMAD 7, pSMAD 3 and
phosphorylation of SMAD 3 did not correlate with the
presence or absence of Merlin in any grade of meningio-
mas. Consequently, Merlin may not regulate TGF-bRs or
SMAD 3 phosphorylation. The role of NF-2 gene expres-
sion and Merlin in the pathogenesis of sporadic meningi-
omas remains uncertain. Moreover, loss of Merlin may not
J Neurooncol (2011) 103:277285
Fig. 3 Effects of TGF-b1 on WHO grade II (a) and grade II (MC5-7)
and III (MC8) meningioma cell proliferation (b). Meningioma cells
were treated with TGF-b1 (10 ng/ml) and or PDGF-BB (10 ng/ml)
for 72 h. CyQUANT analysis of DNA measured by fluorescence at
480/520 nm. Changes in DNA fluorescence correlates with cell
proliferation. TGF-b1 failed to suppress basal proliferation in cells
from all 8 higher grade primary meningioma cultures and PDGF
stimulation of proliferation in 6 of 7 WHO grade II and the grade III
meningioma cells. Bars represent standard errors. * P\ 0.05,
** P\ 0.01, *** P\ 0.005, **** P\ 0.001, ***** P\ 0.0005
correlate consistently with dedifferentiation and progres-
sion of meningiomas [54, 55].
The role of TGF-b in the pathogenesis of meningiomas
remains to be established. Normal leptomeninges appear to
Fig. 4 Western blot analysis of TGF-b1 effects on SMAD 3,
p38MAPK and p44/42 MAPK phosphorylation/activation in menin-
gioma cells cultured from WHO grade II meningiomas. TGF-b1
produced a mild increase in phosphorylation of SMAD 3 in 2 of 4
WHO grade II meningioma cultures which had shown no change in
283
synthesize and secrete all three isoforms of TGF-b [8]. In
combination with TGF-b in CSF, this may exert a chronic
inhibitory or differentiating effect on cells in the lepto-
meninges [8]. Conceivably, this effect continues in nascent,
slow growing foci of meningioma cells and contributes to
the long, minimally symptomatic period in patients
developing these tumors. A reduction in TGF-bRs and/or
sensitivity to TGF-b growth inhibition may occur with
progression [56]. This scenario is seen in astrocytes and
low grade astrocytomas that secrete TGF-b-1 and 2 and are
inhibited by exogenous TGF-b. However, dedifferentiating
astrocytoma cells become progressively resistant to the
growth inhibitory effects of TGF-b as they evolve into
malignant gliomas [57]. Similarly, intestinal epithelium
and benign adenoma cells are also inhibited by TGF-b but
become progressively resistant to TGF-b as adenoma cells
dedifferentiate into carcinomas [5860]. Other studies have
noted loss of sensitivity to TGF-b due to decreased
expression in ovarian or colorectal carcinomas [33, 38] or
mutations in the TGF-bRI genes occurs in primary and
metastatic breast carcinomas [32]. Loss of sensitivity to the
inhibitory effects of TGF-b in meningiomas might also be
downstream from receptor activation via alterations in
SMAD signaling. Moreover, this might be in concert with
other growth factors. For example, activation of mitogenic
EGF receptors (present on meningioma cells) activates
MAPK/Erk kinase which hyperphosphorylates SMAD 2
and 3 preventing their movement into the nucleus and
TGF-b signaling [48]. EGF also increases SMAD 7 which
is inhibitory to SMAD 2/3 signaling [51]. Thus, early in the
development of meningiomas, other factors may slowly
override any tonic inhibitory effects on meningioma cell
proliferation. Studies underway may clarify if, during
dedifferentiation, TGF-bs inhibition of meningioma cell
growth is overridden by other signaling or TGF-b
switches from an inhibitory to stimulatory influence.
In summary, this study found only reduced expression of
TGF-bRIII in a limited number of grade III meningiomas
and reduced sensitivity to TGF-b1 growth inhibition.
DNA synthesis after treatment. TGF-b1 also increased phosphoryla-
tion of p38MAPK in 2 of 4 compared with controls and phosphor-
ylation was detected in p44/42 MAPK in 3 of 5. C control, T TGF-b1,
P PDGF-BB
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Although suggestive, the prevalence and impact of these
findings remains to be established and defects in TGF-bR
III may only occur in a subpopulation of meningiomas.
Nonetheless, restoring or boosting TGF-b inhibition of
meningioma cell proliferation may be an important
objective in the design of new chemotherapies for select
meningiomas.
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