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FEBS Letters 582 (2008) 341–345

Induction of podoplanin by transforming growth factor-b
in human ﬁbrosarcoma
Hiroyuki Suzukia,1, Yukinari Katob,*,1, Mika Kato Kanekob, Yukari Okitaa,
Hisashi Narimatsub, Mitsuyasu Katoaba
Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki 305-8575, Japan
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Open Space Laboratory C-2,
1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan

Received 10 December 2007; revised 13 December 2007; accepted 14 December 2007
Available online 26 December 2007
Edited by Veli-Pekka Lehto

Abstract Podoplanin/aggrus is increased in tumors and its
expression was associated with tumor malignancy. Podoplaninon cancer cells serves as a platelet-aggregating factor, which is
associated with the metastatic potential. However, regulatorsof podoplanin remain to be determined. Transforming growth
factor-b (TGF-b) regulates many physiological events, including
tumorigenesis. Here, we found that TGF-b induced podoplanin inhuman ﬁbrosarcoma HT1080 cells and enhanced the plateletaggregating-ability of HT1080. TGF-b type I receptor inhibitor(SB431542) and short hairpin RNAs for Smad4 inhibited the
podoplanin induction by TGF-b. These results suggest that
TGF-b is a physiological regulator of podoplanin in tumor cells.
 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Podoplanin; Platelet aggregation; TGF-b; Smad;Fibrosarcoma

1. Introduction
A mucin-type transmembrane sialoglycoprotein, podoplanin(aggrus) is highly expressed in lymphatic endothelial cells(LECs) [1]. Several lines of evidence obtained using podoplanin knockout mice suggest that podoplanin is crucially involved in lymphatic vessel formation [2]. Moreover,podoplanin is reportedly expressed in several tumor cells suchas various squamous cell carcinomas, testicular tumors, mesothelioma, and brain tumors, although podoplanin is not expressed in gastrointestinal and pulmonary adenocarcinomas,
which frequently undergo metastasis [3]. Recent investigations
have reinforced the notion that expression of podoplanin is
associated with tumor malignant progression [4].
Podoplanin belongs to the family of type-I transmembranesialomucin-like glycoproteins and possess the platelet-aggregating activity and metastasis-promoting ability [5,6]. The segmentof EDxxVTPG in the extracellular domain, designated as aplatelet-aggregation-stimulating (PLAG) domain, is critical
for the activity of podoplanin. In particular, this motif, which
is highly conserved across species, is triplicated in tandem [7].
In a study of targeted mutagenesis of podoplanin molecules,
we obtained evidence that Thr residues in the PLAG domainplay an important role in platelet aggregation [5]. Recently, we
puriﬁed human podoplanin from the glioblastoma cell line
LN319 cells using an anti-human PLAG domain monoclonal
antibody (NZ-1), and showed that podoplanin possesses a
disialyl-core1 structure at Thr52 in the PLAG domain [8]. Furthermore, C-type lectin-like receptor 2 (CLEC-2), which is a
non-classical C-type lectin, was identiﬁed as an endogenous
receptor of podoplanin on platelet [9]. NZ-1 neutralized theassociation between podoplanin and CLEC-2, and suppressed
podoplanin-induced platelet aggregation and metastasis [10].
Transforming growth factor-b (TGF-b) superfamily of
extracellular growth factors regulates cell proliferation, diﬀerentiation, apoptosis, and morphogenesis. Upon ligand binding
to two diﬀerent types of serine/threonine kinase receptors, type
I receptor is activated by type II receptor and transduces intracellular signals by phosphorylation of receptor-regulated Smad
(R-Smad). Among R-Smad, Smad1, Smad5, and Smad8 arephosphorylated by the bone morphogenetic protein (BMP)
receptors, whereas Smad2 and Smad3 are phosphorylated by
the TGF-b/activin receptors [11]. Phosphorylated R-Smadforms a functional signaling complex with Smad4 and translocates into the nucleus to regulate expression of the ligandresponsive genes [12]. TGF-b regulates a number of genes ina cell type and context dependent manner.
Podoplanin has been reported to be expressed in invading
front of tumors cells and be involved in tumor invasion [13];therefore, some stromal factors might be involved in the induction of podoplanin. Previously, Interkeukin-3 (IL-3) was reported to induce podoplanin in human endothelial cells [14].
However, it has not been clariﬁed how the expression of podoplanin is regulated in tumor cells. In this study, we examined theexpression of podoplanin by TGF-b using various cell lines.

*

Corresponding author. Fax: +81 29 861 3191.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
1

2. Materials and methods

These authors contributed equally to this work.

Abbreviations: PLAG, platelet-aggregation-stimulating; TGF-b, transforming growth factor-b; R-Smad, receptor-regulated Smad; IL,interleukin; EMT, epithelial–mesenchymal transition

2.1. Cells and reagents
HT1080, 293T, HaCaT, and MCF10A cells were obtained from theAmerican Type Culture Collection (ATCC). These cells were cultured
in DulbeccoÕs modiﬁed EagleÕs medium (Sigma, St. Louis, MO)

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2007.12.028

Author's personal copy
342
supplemented with 10% fetal bovine serum (Sigma) and 1% of penicillin–streptomycin solution (Invitrogen Corp., Carlsbad, CA). Puromycin and SB431542 was obtained from Sigma and Calbiochem
Novabiochem Corp. (San Diego, CA), respectively. TGF-b was purchased from R&D Systems (Minneapolis, MN).
2.2. DNA constructs
Sequence of oligonucleotides against human Smad4 cDNA was asfollows: GGATTTCCTCATGTGATCT. The complementary oligonucleotides were annealed and ligated in a pSUPER. puro vector (OligoEngine, Seattle, WA). Stable HT1080 clones transfected with
pSUPER-short hairpin RNAs for Smad4 (shSmad4) were selected
and maintained in the presence of puromycin (1 lg/ml).
2.3. Reverse transcription (RT)-PCR
Total RNA was prepared using Isogen (Nippongene Co. Ltd., Tokyo, Japan). Reverse transcription was performed with Superscript
III (Invitrogen Corp.), and PCR was performed using the primers
listed as follows: podoplanin – 5 0 -CCAGGAGAGCAACAACTCA
A-3 0 (forward), 5 0 -GATGCGAATGCCTGTTACAC-3 0 (reverse);
Smad7 – 5 0 -GGAAGTCAAGAGGCTGTGTT-3 0 (forward), 5 0 -GCT
TTCTCCTCCCAGTATGC-3 0 (reverse); b-actin – 5 0 -CAAGAGATGGCCACGGCTGCT-3 0 (forward), 5 0 -TCCTTCTGCATCCTGTC
GGCA-3 0 (reverse).
2.4. Luciferase assay
Cells were transfected with (CAGA)9 MLP-luc using FuGENE6(Roche Diagnostics K.K., Tokyo, Japan). Luciferase activities were
determined by Luciferase Assay Systems (Promega K.K., Tokyo, Japan) and normalized to b-galactosidase activity of co-transfected
CH110 (GE Healthcare UK Ltd., Buckinghamshire, UK).
2.5. Flow cytometry
Expression levels of human podoplanin were compared for conﬁrmation using ﬂow cytometry. HT1080 cells, which were collected by trypsinEDTA treatment, were incubated with NZ-1 (0.1 lg/ml) for 1 h at 4 C.
Then the cells were incubated with Oregon green-conjugated anti-rat
antibodies (Invitrogen Corp.) for 30 min. Flow cytometry was performed using FACS Caliber (BD Biosciences, Barintree, MA).
2.6. Western blot analysis
The cell lines were solubilized with lysis buﬀer (1% Triton in PBS)
and electrophoresed under reducing conditions on 10–20% polyacrylamide gels. The separated proteins were transferred to a PVDF membrane. After blocking with 4% skim milk in PBS, the membrane wasincubated with NZ-1 (rat, 0.1 lg/ml), anti-Smad2 (mouse, 0.25 lg/
ml; Transduction Laboratories, Lexington, KY), anti-Smad4 (mouse,
0.2 lg/ml; Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-phospho-Smad2 (rabbit) [15], and anti-b-actin antibody (mouse, 0.2 lg/ml;
Sigma), and then with peroxidase-conjugated anti-rat, anti-mouse, or
anti-rabbit antibodies (1/1000 diluted; GE Healthcare UK Ltd.), anddeveloped for 1 min with ECL reagents (GE Healthcare UK Ltd.)
using Kodak X-Omat AR ﬁlm.
2.7. Platelet aggregation assay by WBA Carna
Heparinized human whole blood (WB) was drawn from healthydrug-free volunteers. Platelet aggregation was measured according to
the screen ﬁltration pressure method using WBA Carna (IMI, Saitama,
Japan) [3,8,10]. Two hundred microliters each of human whole blood
samples in four reaction tubes were stirred at 1000 rpm at 37 C andpre-incubated for 1 min, followed by addition of 10 ll each of
HT1080 cells (1 · 105 cells or 4 · 104). Using a 3.7-mm-diameter syringe containing screen microsieves made of nickel, with 300 openingsof 20 · 20 lm2 in a 1 mm-diameter area, WB samples were sucked todetect aggregation pressure at a rate of 200 ll/6.4 s 2 min later. The ﬁnal platelet aggregation pressure of each reaction tube was determinedat the pressure rate (%) of a pressure sensor connected to the syringe.
2.8. Statistical analyses
Results are expressed as the means ± S.D. StudentÕs t-test was used
to determine signiﬁcance among the groups. A value of p < 0.05 wasconsidered signiﬁcant.

H. Suzuki et al. / FEBS Letters 582 (2008) 341–345

3. Results and discussion
3.1. Induction of podoplanin by TGF-b in hunan ﬁbrosarcoma
HT1080 cells
To examine the expression of podoplanin by TGF-b, we performed Western blot analysis using anti-podoplanin monoclonal antibody, NZ-1 [3] in various cell lines. As shown in
Fig. 1A, TGF-b induced podoplanin in human ﬁbrosarcoma
HT1080 cells, but not in breast epithelial MCF-10A cells andhuman keratinocyte, HaCaT cells, which are known to exhibit
the high responsiveness to TGF-b. Although human embryonic kidney 293T cells express low level of podoplanin, theinduction of podoplanin by TGF-b was hardly observed. Timecourse experiment showed that podoplanin mRNA increased
at 12 h treatment with TGF-b (Fig. 1B), followed by the
podoplanin protein at 24 h (Fig. 1C). Induction of Smad7
which is a target gene of TGF-b signaling, were observed in
HT1080 cells, indicating that TGF-b-Smad signaling pathway
is intact in HT1080 cells (Fig. 1B). Phosphorylation of Smad2
by TGF-b was also detected (Fig. 1C). Furthermore, we foundthat SB431542, a selective inhibitor of TGF-b type I receptor,
inhibited the induction of podoplanin by TGF-b in a dose
dependent manner (Fig. 1D). These results indicated that
TGF-b stimulates podoplanin expression in HT1080 cells.
TGF-b is a potent growth inhibitor with tumor-suppressing
activity. Cancer cells often show resistance to this growth inhibition either because of genetic loss of TGF-b signaling components or, more commonly, because of downstream
perturbation of the signaling pathway, such as by Ras activation [16]. Carcinomas often secrete excess TGF-b and respondto it by enhanced invasion and metastasis [17]. TGF-b isknown to induce epithelial–mesenchymal transition (EMT)
through the induction of SIP1 and d EF1 [18], Snail familytranscriptional factors [19], and the suppression of inhibitorof DNA binding (Id) proteins [20]. Furthermore, TGF-b promotes bone metastasis through the induction of IL-11 and connective tissue growth factor [21]. Podoplanin has been also
reported to induce EMT in MDCK cells linked to the activation of RhoA, and increased cell migration and invasiveness[13]. Although TGF-b induces podoplanin in HT1080 cells,
it is not suﬃcient to induce the invasion and migration of
HT1080 cells [22]. Even though, podoplanin might be one ofcancer progressive factor, which is regulated by TGF-b.
3.2. Platelet aggregation by HT1080 cells
To conﬁrm the expression of functional podoplanin on cellsurface, we performed the ﬂow cytometry using anti-podoplanin (NZ-1). As shown in Fig. 2A, the expression of podoplaninon cell surface was observed in the absence of TGF-b and its
expression was further stimulated by TGF-b. We next investigated the platelet aggregation by HT1080 cells in the presenceor absence of TGF-b. Platelet aggregation was observed incontrol HT1080 cells, and was further stimulated by TGF-btreated HT1080 cells (Fig. 2B). These results suggest that functional podoplanin was induced on cell surface of HT1080 cells.
3.3. Eﬀect of Smad4 knockdown on the induction of podoplanin
by TGF-b
We next investigated the involvement of Smad pathway on
the expression of podoplanin by TGF-b. We established
HT1080 cells which stably expressed short hairpin RNAs forAuthor's personal copy
H. Suzuki et al. / FEBS Letters 582 (2008) 341–345

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Fig. 1. Induction of podoplanin by TGF-b in human ﬁbrosarcoma HT1080 cells. (A) HaCaT, 293T, HT1080, and MCF10A cells were treated with
TGF-b (2.5 ng/ml) for 24 h. Western blot analysis was performed using anti-podoplanin antibody (NZ-1). The b-actin protein is used as a loading
control. (B and C) HT1080 cells were treated with TGF-b for indicated time periods. (B) Semi-quantitative RT-PCR analysis was performed to detect
podoplanin and Smad7 mRNA. b-actin is used as internal control. (C) Western blot analysis was performed using NZ-1, anti-phospho-Smad2, antiSmad2, and anti-b-actin antibodies. (D) HT1080 cells were treated with TGF-b in the presence or absence of SB431542 at indicated concentrationsfor 24 h. Western blot analysis was performed using antibodies as indicated. D indicates dimethyl sulfoxide (solvent).

Fig. 2. Cell surface expression of podoplanin and platelet aggregation
by HT1080 cells. (A) Flow cytometric analyses of NZ-1 to HT1080
cells which were treated with TGF-b for 24 h. (B) Platelet aggregation
was measured using WBA Carna with the screen ﬁltration pressure
method. Data are means ± S.D. of three independent experiments.

Smad4 (shSmad4). We obtained two transfectants (clones 5
and 10), and these Smad4 were signiﬁcantly reduced comparedwith control (pSUPER) transfected cells (Fig. 3A). However,
the expression of Smad2 and phosphorylation of Smad2 by
TGF-b were similar between control and shSmad4 transfected
cells. The transcriptional activation of (CAGA)9 MLP-luc by
TGF-b was signiﬁcantly reduced in shSmad4 transfected cells(Fig. 3B). Using these transfectants, we investigated the induction of podoplanin by TGF-b. As shown in Fig. 3C, the induction of podoplanin by TGF-b was blocked in shSmad4transfected cells. These results suggest that TGF-b-Smad pathway is important for the induction of podoplanin by TGF-b in
HT1080 cells.
TGF-b regulates various target genes via Smad pathway[23]. However, podoplanin was not induced by TGF-b in HaCaT and MCF10A cells (Fig. 1A), which suggested that TGFb-Smad signaling alone is not suﬃcient to induce podoplanin.
Therefore, Smad would cooperatively regulate podoplaninwith other transcriptional factors, which maintain the basal
expression of podoplanin in HT1080 cells. Recent report revealed that transcriptional factor Sp1 and Sp3 stimulate basal
podoplanin expression, and methylation status of its promoter
confers cell-type speciﬁc podoplanin expression [24]. Smads
have been shown to interact with Sp1, and regulates various
target genes including p21 [25]. Further study is needed to
identify the transcriptional factors which regulate podoplaninwith Smads.
In conclusion, we found that TGF-b induced podoplanin inhuman ﬁbrosarcoma HT1080 cells. The platelet aggregation
by HT1080 cells was stimulated by treatment of cells with
TGF-b, which may lead to acquirement of high metastatic
ability of cancer cells. Furthermore, we are now investigating
whether podoplanin is induced by TGF-b in other cancer cells.

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344

Fig. 3. Eﬀect of Smad4 knockdown on the TGF-b-induced podoplanin expression. (A) Western blot analysis of Smad4 in shSmad4transfected HT1080 cells (clones 5 and 10). Cells were treated with
TGF-b for 1 h. Western blot analysis was performed using antiSmad4, anti-phospho-Smad2, anti-Smad2, and anti-b-actin antibodies.
(B) (CAGA)9 MLP-luc activation by TGF-b was inhibited in
shSmad4-transfected cells. Cells were transfected with (CAGA)9
MLP-luc for 24 h and treated with TGF-b for another 24 h. Luciferase
activity was determined as described in Section 2. Error bars representmeans ± S.D. (C) Eﬀect of Smad4 knockdown on the TGF-b-induced
podoplanin expression. Cells were treated with TGF-b for 24 h.
Western blot analysis was performed using NZ-1 and anti-b-actin
antibodies.

In the future, podoplanin and TGF-b might represent a promising therapeutic target in cancer invasion and metastasis.
Acknowledgements: We thank Ms. Nana Matsuura for technical assistance. This study was supported in part by grants-in-aid for ScientiﬁcResearch from the Ministry of Education, Culture, Science, Sports andTechnology of Japan (H. Suzuki), by Mitsubishi Pharma Research
Foundation (Y. Kato), by the YASUDA Medical Foundation (Y.
Kato), Inoue Foundation for Science (Y. Kato), and by the Osaka
Cancer Research Foundation (MK. Kaneko).

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