Biochem. J. (2011) 440, 273–282 (Printed in Great Britain)
273
doi:10.1042/BJ20110167
Heparanase enhances nerve-growth-factor-induced PC12 cell
neuritogenesis via the p38 MAPK pathway
Hengxiang CUI*1 , Chenghao SHAO†1 , Qin LIU*, Wenjie YU‡, Jianping FANG*, Weishi YU‡, Amjad ALI‡ and Kan DING*2
*Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, †Department of Surgery, Changhai
Hospital, Shanghai 200433, China, and ‡The Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
Heparanase is involved in the cleavage of the HS (heparan sulfate)
chain of HSPGs (HS proteoglycans) and hence participates in
remodelling of the ECM (extracellular matrix) and BM (basement
membrane). In the present study we have shown that NGF
(nerve growth factor) promoted nuclear enrichment of EGR1
(early growth response 1), a transcription factor for heparanase,
and markedly induced heparanase expression in rat adrenal
pheochromocytoma (PC12) cells. K252a, an antagonist of the
NGF receptor TrkA (tyrosine kinase receptor A), decreased
heparanase protein expression induced by NGF in PC12 cells.
Suramin, a heparanase inhibitor, decreased heparanase in PC12
cells and blocked NGF-induced PC12 neuritogenesis. Stable
overexpression of heparanase activated p38 MAPK (mitogenactivated protein kinase) by phosphorylation and enhanced the
neurite outgrowth induced by NGF, whereas knock down of
heparanase impaired this process. However, overexpression of
latent pro-heparanase with a Y156A mutation still led to
enhanced NGF-induced neurite outgrowth and increased p38
MAPK phosphorylation. Inhibition of p38 MAPK by SB203580
suppressed the promotion of NGF-induced neuritogenesis by the
wild-type and mutant heparanase. The impaired differentiation
by knock down of heparanase could be restored by transfection
of wild-type or mutant heparanase in PC12 cells. The results of
the present study suggest that heparanase, at least in the nonenzymatic form, may promote NGF-induced neuritogenesis via
the p38 MAPK pathway.
INTRODUCTION
endothelial cells and astrocytes [12,16–18]. Heparanase expressed
by these cells is thought to facilitate cellular migration and
invasion, which is associated with autoimmunity, inflammation
and angiogenesis. It is also believed to function as an adhesion
molecule, which mediates cell adhesion to the ECM resulting
in integrin-dependent cell spreading, tyrosine phosphorylation
of paxillin and reorganization of the actin cytoskeleton [19,20],
independent of its endoglucuronidase activity. A previous study
demonstrated that both heparanase mRNA and protein are
also expressed in neuronal cells and glial cells. Heparanase
immunoreactivity was found not only in the cytoplasm, but also
in the nucleus of the neurons [21]. These findings suggest that
heparanase probably plays roles in the normal function of the CNS
(central nervous system) [21], as mobile HS fragments generated
as a result of the enzymatic heparanase activity may facilitate HSbinding growth factor signalling in the regulation of proliferation
and differentiation [22].
Rat adrenal pheochromocytoma (PC12) cells have been
extensively used as a model of catecholaminergic neurons
in culture, as well as for the study of neuronal apoptotic
death. Associated with the NGF (nerve growth factor)-induced
morphological change is the growth of axon-like processes,
called neuritogenesis, which is characterized by growth arrest, the
elaboration of long branching neurites and electrical excitability
[23]. The morphological hallmark of neuronal differentiation
is neurite sprouting, elongation and subsequent maturation of
HSPGs [HS (heparan sulfate) proteoglycans] are ubiquitously
distributed on the cell surface, ECM (extracellular matrix) and
BM (basement membrane) of a wide range of cells in vertebrate
and invertebrate tissues [1,2]. HSPGs consists of a core protein
to which several linear HS GAG (glycosaminoglycan) chains are
covalently O-linked. HSPGs play key roles in the self-assembly
and integrity of the multimolecular architecture of the BM and
ECM [2,3]. Hence, they affect diverse biological processes,
such as cell migration, embryonic morphogenesis, angiogenesis,
metastasis, inflammation, neurite outgrowth and tissue repair. HS
on HSPGs act as co-receptors for growth factors, such as FGF
(fibroblast growth factor) and VEGF (vascular endothelial growth
factor) to influence cell behaviour, including differentiation [4,5].
Theoretically, the HS structural motif modified by its biosynthesis
and/or degradation enzyme may mediate cell differentiation [6–
10]. Heparanase, which is considered to be a dominant endo-β-Dglucuronidase in mammalian tissues and has been identified in a
variety of cell types and tissues, contributes to the cleavage of the
HS chains on HSPGs and hence participates in degradation and
remodelling of the ECM and BM [11,12]. Heparanase activity
is commonly considered to be correlated with the metastatic
ability of various tumour-derived cells [13–15]. In addition to
tumour-derived cells, heparanase is also expressed in normal
cells, including leucocytes such as neutrophils, macrophages,
Key words: heparanase, neurite outgrowth, neuritogenesis, neuron
differentiation, p38 mitogen-activated protein kinase (MAPK).
Abbreviations used: BM, basement membrane; BrdU, bromodeoxyuridine; CNS, central nervous system; DAPI, 4 ,6-diamidino-2-phenylindole; DMEM,
Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; EGR1, early growth response 1; EMSA, electrophoretic mobility-shift assay; ERK,
extracellular-signal-regulated kinase; GAG, glycosaminoglycan; GAP43, growth-associated protein 43; GFP, green fluorescent protein; HEK, human
embryonic kidney; HS, heparan sulfate; HSPG, HS proteoglycan; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK
kinase; M-MLV, Moloney murine leukaemia virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H -tetrazolium bromide; NGF, nerve growth factor; RT,
reverse transcription; SAPK, stress-activated protein kinase; shRNA, short hairpin RNA; VEGF, vascular endothelial growth factor.
1
These authors contributed equally to this work
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
274
H. Cui and others
neurites into axons and dendrites. Phenotypic changes associated
with NGF-induced differentiation include the biosynthesis of
neurotransmitters, the acquisition of electrical excitability, along
with the growth of axon-like extensions during neuritogenesis
[23]. Neuritogenesis is a complex phenomenon that involves
multiple interactions between the growing neurite and the ECM.
Although increased heparanase expression was observed during
NGF-induced neuronal differentiation in PC12 cells, the function
of heparanase in neuritogenesis is not clear [24]. As heparanase
participates in remodelling of the ECM and BM, which are
essential for neuronal differentiation, in the present study we
investigated its role in neuritogenesis using the PC12 model.
EXPERIMENTAL
General materials
NGF, LipofectamineTM 2000 and 1 × DMEM (Dulbecco’s modified Eagle’s medium) were from Invitrogen. Suramin sodium salt,
the TrkA antagonist K252a and MTT [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-2H-tetrazolium bromide] were from Sigma–
Aldrich. The selective p38 MAPK (mitogen-activated protein
kinase) inhibitor (SB203580) was purchased from Calbiochem.
The polyclonal antibody (catalogue number ab59787) against
heparanase was from Abcam. The anti-(p44/42 MAPK)
[ERK1/2 (extracellular-signal-regulated kinase 1/2)] antibody
(catalogue number 9102), anti-(phospho-p44/42 MAPK)
(ERK1/2) (Thr202 /Tyr204 ) antibody (catalogue number 3477), antiSAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal
kinase) antibody (catalogue number 9252), anti-(phosphoSAPK/JNK) (Thr183 /Tyr185 ) antibody (catalogue number 4668),
and monoclonal antibodies against phospho-p38 MAPK
(Thr180 /Tyr182 ) (catalogue number 4511) and EGR1 (early growth
response 1) (catalogue number 4153), and a polyclonal antibody
against p38α MAPK (catalogue number 9212) were purchased
from Cell Signaling Technology. The anti-(histone H1) (catalogue
number sc-8030) antibody was from Santa Cruz Biotechnology.
BrdU (bromodeoxyuridine), rabbit anti-actin antibody, antiBrdU monoclonal antibody and FITC-conjugated goat antimouse antibody were from Sigma–Aldrich. The pMD-18T easyclone vector was from TaKaRa Biotechnology. IRDye® 800CWconjugated goat (polyclonal) anti-rabbit IgG (catalogue number
926-32211) and IRDye® 800CW-conjugated goat (polyclonal)
anti-mouse IgG (catalogue number 926-32210) were from LICOR Biosciences. The M-MLV (Moloney murine leukaemia
virus) reverse transcriptase, endonucleases and T4 ligase were
from TaKaRa Biotechnology.
Cell culture and stable transfections
PC12 cells were obtained from the American Type Cell Culture
collection. Native PC12 cells were maintained in DMEM
(Invitrogen) supplemented with 10 % heat-inactivated horse
serum (Invitrogen) and 5 % FBS (fetal bovine serum, Invitrogen)
at 37 ◦ C in a humidified atmosphere containing 5 % CO2 .
LipofectamineTM 2000 (Invitrogen) was used for transfections
according to the manufacturer’s recommendations. The stably
transfected PC12 cells were screened under neomycin selection.
Briefly, after the vectors were transfected into PC12 cells using
the LipofectamineTM 2000 reagent, the cells were diluted at a ratio
of 1:20 before being selected with 800 μg/ml neomycin (G-418).
After 4 weeks under selection, independent colonies each grown
from one cell were obtained by limiting dilution. Western blot
analysis was used to confirm the stable expression of heparanase
in the cells using a heparanase-specific antibody.
c The Authors Journal compilation c 2011 Biochemical Society
BrdU incorporation and MTT assay
Cells were incubated in the presence of 100 μM BrdU (Sigma–
Aldrich) for 1 h after the cells were treated with or without
suramin for 24 h or 72 h. The cells were then fixed with
4 % paraformaldehyde and treated with 2 M HCl containing
1 % Triton X-100. The cells were stained with an anti-BrdU
monoclonal antibody (Sigma–Aldrich) and then stained with
an FITC-conjugated goat anti-mouse antibody and DAPI (4 ,6diamidino-2-phenylindole). The green immunofluorescence,
indicating BrdU staining, the total number of DAPI-positive cells,
as well as BrdU-labelled cells, was counted in five to ten different
fields of each well. In the MTT assay, 1 × 104 PC12 cells were
treated with or without suramin for 72 h before being washed
three times with PBS. MTT was then added to the cells at a final
concentration of 0.5 mg/ml and incubated for 4 h. Finally, 100 μl
of DMSO was added followed by an absorbance measurement at
570 nm using a Universal Microplate Reader (Bio-Tek).
Generation of heparanase and mutant heparanase expression
constructs
The human HPSE (GenBank® accession number NM_006665)
gene encoding heparanase was cloned using a PCR-based method.
Briefly, total RNA from HEK (human embryonic kidney)-293
cells was reverse-transcribed into first strand cDNA with MMLV reverse transcriptase. Subsequently, the CDS fragment of
HPSE was amplified by overlapping PCR with PrimeSTAR HS
DNA Polymerase (TaKaRa). The following primers were used:
forward primer 5 -TATAGAATTCACCATGGTCCTGCGCTCG
AAGC-3 (A1), and reverse primer 5 -CTCCTGGTAGGGCCATTCCAACCGTAA-3 (A2); and forward primer 5 -GAGCCCTCGTTCCTGTCCG-3 (B1), and reverse primer 5 -CCTCTAGACTGATGCAAGCAGCAACTTTGGCATT-3 (B2). Underlining
indicates the specific sequences recognized by restriction
endonucleases which were used for DNA construction. First, PCR
was performed using primers A1 and A2 to obtain fragment A,
and then performed with primers B1 and B2 to produce fragment
B. A second PCR with primers A1 and B2 was then used to obtain
the full-length clone using fragments A and B as a template
after gel-purifying extraction (the molar ratio of A and B was
1:1). After digesting with the endonucleases EcoRI and XbaI, the
amplified product of 1652 bp was inserted into pCDNA3.1/mycHisB (Invitrogen) with T4 ligase, forming pCDNA3.1/myc-HisBhHPSE which was the final heparanase expression plasmid with
Myc and His tags fused to the C-terminus. This construct was
sequenced by Sangon Biological Engineering Technology &
Services (Shanghai, China). After transfection of PC12 cells with
this construct, three stable cell lines were obtained after selection
by neomycin and designated 9406, 9415 and 9416.
Since substitution of the conserved tyrosine residue at position
156 with alanine has been shown to abolish normal processing and
activation of pro-heparanase [25], a Y156A mutant heparanase
was constructed by site-directed mutagenesis. First, the fulllength wild-type HPSE fragment from pCDNA3.1/myc-HisBhHPSE was inserted into pLVX-IRES-ZsGreen1 at the EcoRI
and XbaI sites to obtain the pLVX-IRES-ZsGreen1-HPSE vector
(ZS-HPSE). In ZS-HPSE, there are two BamHI digestion sites.
One is at nt 389 of the full-length wild-type HPSE (position
‘A’ in the start codon ‘ATG’ was counted as the first base). The
other is at nt 8 after the XbaI site within the backbone vector
pLVX-IRES-ZsGreen1. Secondly, the mutant HPSE fragment,
corresponding to nt 390–1629, also containing two BamHI digestion sites, was substituted into ZS-HPSE at the two BamHI sites.
The mutant HPSE fragment was amplified by two-step PCRs.
Heparanase augments neuritogenesis
The first-step PCR was conducted using the primer pairs HPSEMUT-STEP1-F, 5 -gagaagttacggtTGGAATGGCCCTACCAGGAGCAATTGCTACTCCGAGAACACgagCA-3 , and BamH1HPSE-R, 5 -GGATCCTCAGATGCAAGCAGCAACTTTG. gag
is the mutated site designed in the primer. The second-step
PCR, using the PCR product after gel-purifying extraction
of the first-step PCR as a template, was performed with the
primers BamH1-389-STEP2-F, 5 -CAAATATGGATCCATCCCTCCTGATGTGGAGgagaagttacggt-3 and BamH1-HPSE-R
(primer sequence described above). The bold lowercase sequences
in the two sense PCR primers are the overlapping bases.
Underlining indicates the specific sequences recognized by
restriction endonucleases which were used for DNA construction.
The PCR product of the second step contains the mutant HPSE
fragment (from 390 to 1629) and was subcloned into the pMD-18T
vector before being sequenced and digested with the endonuclease
BamHI. Subsequently, the BamHI-digested fragment obtained
after gel-purifying extraction was substituted into ZS-HPSE at
the two BamHI sites, obtaining pLVX-IRES-ZsGreen1-M-HPSE
(ZS-M-HPSE).
Knock down of the heparanase gene by shRNA (short hairpin RNA)
The heparanase shRNA expression vector pGCsi-U6/Neo/GFP
(GFP is green fluorescent protein) was designed and constructed
by the Genechem Company. The target sequence is 5 -ACCTCCATAATGTCACCAA-3 , with the loop designed as TTCAAGAGA. The shRNA vector was transfected into PC12 cells using
LipofectamineTM 2000 according to the manufacturer’s protocol.
The empty vector served as a negative control. After transfection
of PC12 cells with the shRNA vector and selection with neomycin,
three stable cell lines, Sh-2, Sh-7 and Sh-12, were obtained and
confirmed by RT (reverse transcription)–PCR and Western blot
analysis (Figures 2A and 2B).
Lentivirus-based mutant heparanase overexpression
Virus carrying mutant heparanase was produced by transfecting
HEK-293T [HEK-293 cells expressing the large T-antigen
of SV40 (simian virus 40)] cells with pLVX-IRES-ZsGreen1
(ZS), pLVX-IRES-ZsGreen1-HPSE (ZS-HPSE) or pLVX-IRESZsGreen1-M-HPSE (ZS-M-HPSE) with viral packaging vectors
(psPAX2, pMD2G) using a standard calcium phosphate transfection method. Viruses were harvested from the supernatant at
48 h post-transfection and concentrated by ultracentrifugation
at 35 000 rev./min before infecting 2 × 105 PC12 cells with 1 × 107
TCID50. The cells were used for neuritogenesis assays or Western
blot analysis after they were cultured in complete medium without
virus for 24 h.
RT–PCR
Total RNA was isolated using TRIzol® reagent (Invitrogen).
RT was performed with random hexamers as primers. The
final reaction volume was 20 μl, with 1 μg of total RNA from
each cell sample. The reaction mixture contained 0.5 mM
of each dNTP, 2 units/μl RNase inhibitor, 3 mM MgCl2 ,
10 units/μl M-MLV (AMV) reverse transcriptase and 150 ng of
random hexamers, following the manufacturer’s protocol. The
following specific PCR primers were used for amplification: rat
heparanase sense, 5 -CAAGAACAGCACCTACTCACGAAGC3 ; rat heparanase antisense, 5 -CCACATAAAGCCAGCTGCAAAGG-3 ; human heparanase sense, 5 - GCTACTCCGAGAACACTACCAGA-3 ; human heparanase antisense, 5 -TGAATCAATCACTTCTCCACCAG-3 ; β-tubulin III sense, 5 -GGAAC-
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ATAGCCGTAAACTGC-3 ; β-tubulin III antisense, 5 -TCACTGTGCCTGAACTTACC-3 ; GAP43 (growth-associated protein 43)
sense, 5 -TGCTGTGCTGTATGAGAAGAACC-3 ; GAP43
antisense, 5 -GGCAACGTGGAAAGCCGTTTCTTAAAGT-3 ;
18S rRNA sense, 5 -GTGGAGCGATTTGTCTGGTT-3 ; and 18S
rRNA antisense, 5 -CGCTGAGCCAGTCAGTGTAG-3 . PCR
amplification was conducted using the following conditions:
denaturation for 2 min at 94 ◦ C, and 94 ◦ C for 50 s, annealing for
50 s at 55 ◦ C, and extension for 1 min at 72 ◦ C (28 cycles for 18S
rRNA and 30 cycles for human and rat heparanase, β-tubulin III
and GAP43).
Western blot analysis
Cell extracts were prepared by lysis of (1–5) × 106 cells in 50–
200 μl of RIPA buffer [25 mM Tris/HCl (pH 7.4), 150 mM KCl,
5 mM EDTA, 1 % Nonidet P40, 0.5 % sodium deoxycholate
and 0.1 % SDS] containing 1 mM PMSF and a protease
inhibitor cocktail (Sigma–Aldrich) on ice for 30 min, followed
by removal of DNA and cell debris by centrifugation at
12 000 g for 5 min at 4 ◦ C. The resulting supernatants were
collected and frozen at − 80 ◦ C or used immediately. Protein
concentrations were measured with the DC Protein Assay Kit
(Bio-Rad) following the manufacturer’s recommendations. Then,
50 μg of protein/lane were separated by SDS/PAGE (10 %
gels) (unless specified otherwise), transferred on to PVDF or
nitrocellulose membranes (Bio-Rad) and immunoblotted with
antibodies. Specific peroxidase-conjugated secondary antibodies
or IRDye® 800CW-conjugated goat (polyclonal) anti-mouse
(or rabbit) IgG were used to detect protein expression with
an enhanced chemiluminescence kit (Pierce) or the Odyssey
infrared imaging system (LI-COR Biosciences). Nuclear protein
extraction and EMSAs (electrophoretic mobility-shift assays)
were conducted as described previously [26]. Briefly, after NGF
(50 ng/ml) treatment, the cells were harvested by scraping and
resuspending in 25 mM Hepes, 1.5 mM EDTA and 1 mM DTT
(dithiothreitol) (pH 7.6), and homogenized with 10 % glycerol.
The cell lysates were then centrifuged at 4 ◦ C, and protein
concentrations of the nuclear extracts were then determined using
a BCA (bicinchoninic acid) assay. Aliquots of nuclear protein
were then frozen and stored at − 80 ◦ C until use.
Neuritogenesis assay
In neuritogenesis experiments, cells were seeded on 24-well plates
(Corning) at a density of 2 × 103 cells/well. After incubation
overnight, the cells were treated with medium containing 0.5 %
horse serum and 25 ng/ml 2.5S NGF (Invitrogen). Morphology
of PC12 cells treated with NGF (25 ng/ml) or NGF plus the p38
MAPK inhibitor SB203580 (20 μM) for 72 h was evaluated by
counting the proportion of cells containing at least one neurite
that was twice as long as the diameter of the cell body. Neurite
outgrowth was observed with nine random images captured per
well, using either an inverted fluorescence microscope (cells) or
a phase-contrast microscope. The images were captured by a
digital camera at ×200 magnification. All cells in each image
were analysed, with over 200 cells assessed per cell culture plate.
The experiments were repeated at least three times. Mean and
S.D. values were determined for each treatment.
Statistical analysis
Results are expressed as the means +
− S.E.M. The data were
analysed using Student’s t test. P values of <0.05 indicated
significant differences (* P < 0.05; ** P < 0.01).
c The Authors Journal compilation c 2011 Biochemical Society
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Figure 1
H. Cui and others
NGF induced heparanase mRNA expression, whereas heparanase inhibitor blocked neuritogenesis induced by NGF
NGF was used at 50 ng/ml. (A) Heparanase (Hepa) expression in PC12 cells treated with or without NGF for 16 h was analysed by RT–PCR with 18S rRNA as the internal control. (B) Heparanase
proteins were detected in cell extracts of PC12 cells treated with or without NGF for 0, 16, 24, 48 or 72 h by Western blot analysis with β-actin as a loading control. (C) Heparanase proteins were
detected in PC12 cells treated with or without NGF combined with ( + ) or without ( − ) K252a (100 nM) by Western blot analysis. (D) Nuclear protein extracts of PC-12 cells treated with NGF for
0–8 h were analysed by Western blot with anti-EGR-1 and anti-(histone H1) antibodies. (E) Neuritogenesis of PC12 cells treated without or with NGF only or with NGF and suramin (20 μM) for 72 h
under a phase-contrast microscope (×200 maginfication). The arrow indicates a typical neurite at least twice as long as the diameter of the cell body from the differentiated PC12 cell. (F) The effect
of suramin (20 μM) on heparanase expression was measured by Western blot analysis in the presence ( + ) or absence ( − ) of NGF (25 ng/ml). (G) PC12 cell viability was detected by MTT assay
after treatment with or without suramin at 20 μM or 200 μM for 72 h, with the viability of untreated cells set to 100 %. (H) BrdU incorporation into the cell DNA was also used to evaluate the effect
of suramin on PC12 cell viability. Cells were incubated with 100 μM BrdU for 1 h after treatment with or without suramin for 72 h. BrdU-labelled cells, as percentages of total DAPI-positive cells,
were determined by counting five to ten different fields of each well after immunofluorescent staining as described in (I). Data are represented as means +
− S.D. in (G) and (H). (I) Immunofluorescent
images of PC12 cells treated as described in (H) were fixed and probed with an anti-BrdU monoclonal antibody before being detected with an FITC-conjugated goat anti-mouse secondary antibody
and stained with DAPI.
RESULTS
NGF-induced heparanase expression in PC12 cells is attenuated by
the heparanase inhibitor suramin
Heparanase mRNA and protein are expressed in neuronal cells
and glial cells [22], implying that heparanase may play roles in
the normal function of the CNS [22,24]. We used a conventional
neuronal cell model, the PC12 cell line responsive to NGF, to
study its role in neural cell apoptotic death, proliferation and
differentiation [24].
After treatment with NGF (50 ng/ml) for 16 h, heparanase
mRNA was notably up-regulated in PC12 cells (Figure 1A),
consistent with previously published results [24]. NGF also
c The Authors Journal compilation c 2011 Biochemical Society
increased heparanase protein expression in PC12 cells in a timedependent manner (Figure 1B). It was reported that NGF at
least binds to its two receptors, p75NTR and TrkA [27]. However,
only the activated form of TrkA, but not that of p75NTR , is
significantly co-localized with heparanase [28]. This suggests
that TrkA is probably involved in heparanase expression. To
investigate whether the heparanase expression correlated with
the NGF receptor, K252a, an antagonist of TrkA [29], was used
to evaluate the effect of NGF on heparanase expression. K252a
(100 nM) inhibited the heparanase expression induced by NGF,
indicating the involvement of TrkA signalling (Figure 1C). It
is known that NGF binding of TrkA leads to the activation
of ERK signalling and rapid recruitment of EGR1 [30], a
Heparanase augments neuritogenesis
nuclear transcription factor for heparanase in PC12 cells [31,32].
Indeed, EGR1 was dramatically recruited into the nucleus when
induced by NGF in PC12 cells, as demonstrated by Western
blot analysis (Figure 1D) and EMSA (Supplementary Figure S1
at http://www.BiochemJ.org/bj/440/bj4400273add.htm). These
results are consistent with those reported previously [33]. Thus
we deduced that the NGF induction of heparanase in PC12 cells
was likely to be mediated by the TrkA-EGR1 pathway.
As NGF promotes PC12 cell neuritogenesis, we hypothesized
that the increased expression of heparanase may contribute to
the process of neurite outgrowth. Therefore suramin, a chemical
inhibitor of heparanase, was used to explore the role of heparanase
in neuritogenesis. PC12 cells were incubated with NGF in the
presence or absence of suramin (20 μM) for 72 h. We found
that suramin nearly completely blocked neuritogenesis induced
by NGF (50 ng/ml) in PC12 cells compared with the control
group (Figure 1E). Further investigation revealed that suramin
decreased the heparanase expression in PC12 cells treated with
NGF for 24 h (Figure 1F). To confirm that the inhibition of
neuritogenesis by suramin was not due to cell toxicity, the
effect of suramin on PC12 cell viability was tested using the
MTT assay and BrdU incorporation method. In the MTT assay,
suramin, even at a concentration of 200 μM, did not significantly
reduce cell viability after treatment for 72 h (Figure 1G).
Consistently, suramin also did not affect BrdU incorporation
into DNA in PC12 cells after treatment at both concentrations
of 20 μM and 200 μM (Figures 1H and 1I respectively) for
72 h. Therefore the inhibition of NGF-induced neuritogenesis by
suramin observed at 20 μM in PC12 cells was unlikely to be due to
any effect on cell viability. Because suramin as an inhibitor lacks
appropriate specificity for heparanase activity, it could potentially
affect heparanase expression through inhibiting the NGF-induced
PC12 neuritogenesis. Taken together, these results implicated
heparanase in the process of neuritogenesis.
Knock down of heparanase expression impairs NGF-induced
neuritogenesis of PC12 cells
To further determine whether heparanase is required for the
promotion of neuritogenesis by NGF, we then knocked down
the expression of heparanase using shRNA. To achieve this,
PC12 cells were stably transfected with heparanase shRNA. The
heparanase expression at the mRNA (Figure 2A) and protein
(Figure 2B) levels were clearly knocked down in three heparanase
shRNA lines (Sh-2, Sh-7 and Sh-12). The expression of the proheparanase protein (65 kDa) in the above three shRNA cell lines
was lower than that in the negative control cells and sham cells
(Figure 2B).
We further examined the effect of knock down of heparanase
on the NGF-induced neurite outgrowth in PC12 cells using
the Sh-2, Sh-7 and Sh-12 clonal cell lines. These stable
heparanase shRNA cells incubated with NGF for 72 h had
reduced early neurite extension compared with the wildtype counterpart and vector control (Supplementary Figure S2
at http://www.BiochemJ.org/bj/440/bj4400273add.htm). Evaluation of the morphology revealed that the proportion of cells
bearing at least one neurite twice as long as the diameter of
the cell body in the three cell clones was significantly less
than that of the control group and sham group (Figure 2C).
Furthermore, the rescue experiment showed that the impaired
differentiation induced by NGF of cells with knock down of
heparanase was restored by transfection of heparanase (wild-type
or its enzymatically inactive mutant) into these cells (Figures 2D
and 2E). On the basis of these results, we deduced that heparanase
277
(or perhaps the pro-heparanase), at least in part, played a positive
role in NGF-induced PC12 cell neuritogenesis.
Overexpression of heparanase enhances NGF-induced PC12 cell
neuritogenesis
Since NGF-induced PC12 neuritogenesis was attenuated by
knock down of the heparanase gene, we further evaluated
how overexpression of heparanase would affect this process
by generating the construct pcDNA3.1/myc-HisB-hHPSE. PC12
cells were stably transfected with this heparanase overexpression
plasmid using the same method as described in the construction
of the knock down cell clones, which ultimately resulted in
three cell clones, designated 9406, 9415 and 9416. Expression
of heparanase at the mRNA (Figure 3A) and protein (Figure 3B)
levels in the three chosen clones was much higher than in the
control and vector sham control cells when detected using an
antibody against heparanase or the Myc tag (results not shown),
indicating that heparanase was stably overexpressed in these cells.
Neurite outgrowth in the heparanase-overexpressing PC12
cells after stimulation with NGF for 72 h was indeed enhanced
compared with the control groups (Supplementary Figure
S3 at http://www.BiochemJ.org/bj/440/bj4400273add.htm). Morphology analysis showed that the proportion of cells bearing at
least one neurite twice as long as the diameter of the cell body
in the three cell clones was significantly greater than that of the
control groups (Figure 3C). On the basis of the results above, we
suggest that heparanase (or perhaps pro-heparanase) enhanced
NGF-induced PC12 neuritogenesis.
p38 MAPK phosphorylation is required for neuritogenesis
promoted by heparanase
In order to uncover the mechanism underlying the enhancement of
neuritogenesis by heparanase, we evaluated the phosphorylation
levels of p38 MAPK, ERK and JNK in all of the stable
cell lines generated above. Phosphorylation of p38 MAPK
was induced in the cell lines stably expressing heparanase
(Figure 4A and Supplementary Figure S4C at http://www.
BiochemJ.org/bj/440/bj4400273add.htm), whereas it was
impaired in those with heparanase stably silenced (Figure 4B
and Supplementary Figure S4D). However, there was no obvious
change in the phosphorylation levels of ERK and JNK in those
stable cells (Supplementary Figures S4A and S4B respectively).
The results suggested that phosphorylation of p38 MAPK
may be involved in NGF-induced PC12 cell neuritogenesis
augmented by heparanase. To test this hypothesis, PC12 cells
stably overexpressing heparanase were treated with the p38
MAPK phosphorylation inhibitor SB203580 (20 μM) for 72 h,
followed by analysis of p38, ERK and JNK MAPK signalling
components by Western blot. Indeed, although p38 MAPK
phosphorylation was induced by heparanase, this effect was
completely blocked by SB203580 (Figure 4C), whereas ERK
and JNK phosphorylation were not disturbed by this inhibitor
(Supplementary Figure S4E). To explore further the role of the
p38 MAPK pathway in the heparanase enhancement effect on
NGF-induced neurite outgrowth of PC12 cells, neuritogenesis
was examined using the p38 MAPK phosphorylation inhibitor
SB203580. The promoting effect of heparanase on NGF-induced
PC12 cell neuritogenesis was blocked when the cells were treated
with 20 μM SB203580 (Figures 4D and 4E). From these results,
we deduced that heparanase could play a positive role in NGFinduced PC12 neuritogenesis through the p38 MAPK pathway.
c The Authors Journal compilation c 2011 Biochemical Society
278
Figure 2
H. Cui and others
Knock down of heparanase expression led to arrest of neuritogenesis induced by NGF
(A) mRNA levels of heparanase (Hepa) were detected by RT–PCR in cells stably transfected with three clonal PC12 cell lines carrying shRNA against heparanase (Sh-2, Sh-7 and Sh-12), control
cells (Con) and vector control cells (GFP). 18S rRNA was used as the internal control. (B) Heparanase protein expression was detected by Western blot analysis in Sh-2, Sh-7 and Sh-12 cell
lines, vector control (GFP) and control (Con) cells probed with an anti-heparanase polyclonal antibody. Arrows indicate the molecular mass of pro-heparanase (65 kDa) and the active form of
the enzyme (50 kDa). (C) Histogram showing the proportion of cells bearing one neurite at least twice the length of the diameter of the cell body in the neuritogenesis assay. The three cell
clones (Sh-2, Sh-7 and Sh-12), control cells (Con) and vector control (GFP) cells were seeded in 24-well plates at 2 × 103 cells/well and incubated overnight, followed by exposure to medium
(0.5 % horse serum) containing 25 ng/ml NGF for 72 h. Neurite outgrowth was observed with nine random fields per well, and the captured images are shown in Supplementary Figure S2 (at
http://www.BiochemJ.org/bj/440/bj4400273add.htm). All cells in each image were analysed, with over 200 cells assessed per cell culture plate. Results are represented as means +
− S.D. for each
treatment, and the experiments were repeated at least three times. **P < 0.01 and *P < 0.05 for Sh2, Sh7, and Sh12 compared with GFP, by Student’s t test. (D) Histogram showing the proportion
of cells bearing one neurite at least twice the length of the diameter of the cell body in each group in the rescue experiment. (E) Morphology images of neurite outgrowth in the rescue experiment are
shown. In the rescue experiment, Sh-2 was transiently transfected with control vector pLVX-IRES-ZsGreen1 (ZS) (Sh-2 + ZS), vector containing wild-type heparanase pLVX-IRES-ZsGreen1-HPSE
(Sh-2 + ZS-HPSE) or vector subcloned with mutant heparanase pLVX-IRES-ZsGreen1-M-HPSE (Sh-2 + M-HPSE) lentiviral vectors as described in the Experimental section in the presence ( + ) or
absence ( − ) of NGF (25 ng/ml) followed by morphological evaluation. The vector control of the Sh-2 (pGCsi-GFP) stably transfected PC12 cells (GFP) were transfected with pLVX-IRES-ZsGreen1
to serve as a control group. The neuritogenesis assay was performed as described in (C).
To further confirm the role of heparanase in the NGF-induced
neuritogenesis in PC12 cells, the expression of the differentiation
markers β-tubulin III and GAP43 was detected by RT–PCR in the
presence of NGF in the heparanase stable cell lines. After NGF
was added to the culture medium for 16 h, the mRNA expression
levels of β-tubulin III and GAP43 were significantly higher in
cells with overexpression of heparanase, whereas their expression
was significantly lower in cells with heparanase stably silenced
compared with the control and corresponding vector control cells
(Figure 4F and Supplementary Figure S4F). These data suggest
that heparanase may promote the differentiation induced by NGF.
Enzymatically inactive mutant heparanase enhances NGF-induced
neuritogenesis through p38 MAPK phosphorylation
To understand whether the latent form of heparanase (65 kDa)
or the enzyme-active form (50 kDa) was responsible for
differentiation of PC12 cells induced by NGF, an heparanase
Tyr156 to alanine mutant was created and transfected into PC12
c The Authors Journal compilation c 2011 Biochemical Society
cells. Indeed, the latent heparanase mutant was highly expressed
in PC12 cells (Figure 5A). In addition, only p38 phosphoryation of
the components (ERKs, JNK and p38) of the MAPK pathway was
induced by the mutant (Figure 5B). Because only pro-heparanase
proteins (65 kDa) were notably increased in the 9406, 9415 and
9416 stable cells (Figure 3B) and decreased in the stable knockdown cells (Figure 2B), we further overexpressed the latent
heparanase mutant to evaluate its effect on NGF-induced PC12
cell neuritogenesis. The results showed that forced expression of
the pro-heparanase mutant in PC12 cells also enhanced the NGFinduced neurite outgrowth (Figure 5C and Supplementary Figure
S5 at http://www.BiochemJ.org/bj/440/bj4400273add.htm). It
seems that this effect was also dependent on p38 MAPK
phosphorylation, since 20 μM SB203580 blocked the promotion
of the mutant heparanase on NGF-induced PC12 cell neuritogenesis (Figure 5C and Supplementary Figure S5). The proportion
of cells bearing at least one neurite twice as long as the
diameter of the cell body in the cells which overexpressed
the mutant pro-heparanase was significantly greater than that in
Heparanase augments neuritogenesis
Figure 3
Heparanase promoted neuritogenesis induced by NGF
(A) Human heparanase (Hepa) mRNAs were detected by RT–PCR in wild-type (Con) cells, vector
sham control (pcDNA3.1) cells and in three PC12 cell clones (9406, 9415 and 9416) stably
transfected with human heparanase. (B) Heparanase protein expression in wild-type cells, vector
sham control cells and the three chosen clones were determined by Western blot analysis using
a polyclonal anti-(human heparanase) antibody. Arrows indicate the pro-heparanase (65 kDa)
and enzyme-active (50 kDa) forms. (C) Histogram showing the proportion of cells bearing at
least one neurite twice as long as the diameter of the cell body in the 9406, 9415 and 9416
clones, wild-type (Con) and vector sham control (pcDNA3.1) cells. Heparanase-overexpressing
cells, control cells and sham cells were seeded at 2 × 103 cells/well and then treated with NGF
and evaluated for morphology as described in Figure 2. Data are represented as means +
− S.D.
for triplicate treatments, and the experiments were repeated at least three times. The data were
analysed by Student’s t test. *P < 0.05, 9406 compared with pcDNA3.1; **P < 0.01, 9415
compared with pcDNA3.1; *P < 0.05, 9416 compared with pcDNA3.1. Morphology images are
shown in Supplementary Figure S3 at http://www.BiochemJ.org/bj/440/bj4400273add.htm.
the control groups (Figure 5C). Because suramin, the enzyme
inhibitor of heparanase, blocked neuritogenesis induced by NGF,
and the latent heparanase mutant was still able to augment
such NGF-induced neuritogenesis, this finding suggested that
heparanase promotes neuritogenesis at least in PC12 cells
independently of its enzymatic activity. This finding may be
partially explained by the fact that heparanase expression as both
the latent (65 kDa) and active enzymatic forms (50 kDa) was
inhibited by suramin (Figure 1F).
DISCUSSION
Heparanase, commonly used as a HS-degrading enzyme, is
considered to function in acidic environments of the ECM
to facilitate tumour metastasis, angiogenesis and inflammation
[34]. Apart from the well-studied catalytic feature of the
enzyme, heparanase has also demonstrated functions apparently
independent of its enzymatic activity. Non-enzymatic heparanase
enhances cell adhesion [19,20,35,36] and induces p38 MAPK and
Src phosphorylation [36] associated with induction of VEGF [37]
and tissue factor [38] genes.
279
Although heparanase is often considered to regulate autoimmunity, inflammation and angiogenesis, the role of heparanase
in normal development and tissue remodelling has not been wellinvestigated. To date, cell-surface heparanase has been implicated
both in cell migration at early stages of embryogenesis and in
subsequent morphogenesis of the cardiovascular and nervous
systems in the chick embryo [39]. Similarly, knock down of both
XHpaL and XHpaS (heparanase genes in Xenopus), results in the
failure of Xenopus embryogenesis to proceed [6]. Moretti et al.
[40] reported that augmented heparanase activity in the human
olfactory epithelium may represent a physiological mechanism
involved in neural cellular differentiation. In addition, it is of note
that heparanase protein is detected in the dendrites of pyramidal
neurons in the CA1 area of the brain, which indicates that
heparanase might be implicated in the morphological maturation
of spines [41].
In the present study, we first report that heparanase enhanced
neuritogenesis independently of its enzymatic activity in the PC12
cell model, which has been widely used to investigate the cellular
and molecular mechanisms underlying neuronal differentiation
[23], because it alters in morphology into neuron-like cells and
extends long and branching neuritis in response to NGF. We found
that NGF mainly increased latent heparanase (Figures 1B, 1C
and 1F) in PC12 cells, and the latent heparanase mutant also promoted neuritogenesis induced by NGF. This observation indicated
that non-enzymatic heparanase has a predominant enhancing role
in neuritogenesis in PC12 cells induced by NGF, which is distinct
from that reported by Navarro et al. [24]. The results of the present
study enrich the role of heparanase [19] and further pave the way
for studies focusing on non-enzymatic activities of the heparanase
molecule. Neurite outgrowth is a fundamental event in the development of the brain, as well as in the regeneration of damaged
nervous tissue [42]. Cell-adhesion molecules, such as NCAM
(neuronal cell-adhesion molecule) [43] and cadherins [44], transfer signals from the extracellular space which regulate the growth
of the neurite. The non-enzymatic heparanase was reported to mediate cell attachment by recruiting paxillin with increased tyrosine
phosphorylation [19], which has been linked to a remodelling of
the actin cytoskeleton that leads to cell spreading and neurite formation [45]. Thus non-enzymatic heparanase may, as an adhesion
molecule, modulate neuritogenesis signals from the extracellular
space. The present study revealed that NGF also moderately
increased enzymatic heparanase in PC12 cells (Figures 1B and
1C). The enzymatic heparanase can release GAG fragments from
HSPGs [9], which was reported to promote neuritogenesis in vitro
and stimulate nerve re-growth and muscle re-innervations [46,47].
Although enzymatically inactive mutant heparanase can still promote NGF-induced neuritogenesis (Figure 5C and Supplementary
Figure S5), we cannot exclude the role for enzymatic heparanase
in NGF-induced PC12 neuritogenesis. This suggestion is based
on the experimental facts that (i) NGF not only induces latent
heparanase expression, but also mildly increases enzymatic heparanase expression; (ii) forced latent heparanase expression is associated with expression of its enzymatic active form (Figure 3B),
whereas knockdown of latent heparanase by siRNA (small
interfering RNA) also slightly decreases enzymatic heparanase
expression in PC12 cells; (iii) overexpressed mutant enzymatic
heparanase enhances an heparanase enzymatic active form expression in PC12 cells (Figure 5A); and (iv) suramin dramatically
disrupted PC12 neuritogenesis induced by NGF (Figure 1E) and
also inhibited expression of both enzymatic and non-enzymatic
heparanase (Figure 1F), indicating that this protein may promote
NGF-induced PC12 differentiation as a zymogen and in an active
form. All of these results suggest that heparanase augmentation of
neuritogenesis induced by NGF might be through both the
c The Authors Journal compilation c 2011 Biochemical Society
280
Figure 4
H. Cui and others
p38 MAPK phosphorylation was required for heparanase to augment neuritogenesis induced by NGF
Phospho-p38 MAPK (Thr180 /Tyr182 ) was detected by Western blot analysis in human heparanase-overexpressing (9406, 9415 and 9416) (A) and stably silenced (Sh-2, Sh-7, Sh-12) (B) PC12
cell clones compared with wild-type (Control in A and B) and vector control cells (pcDNA3.1 in A; GFP in B) respectively. Total p38 MAPK and β-actin proteins were used as loading controls.
Phosphorylation of p38 MAPK normalized to total p38 MAPK in (A) and (B) is summarized in Supplementary Figure S4C and S4D (at http://www.BiochemJ.org/bj/440/bj4400273add.htm)
respectively. (C) Phospho-p38 MAPK (Thr180 /Tyr182 ) was detected by Western blot analysis in heparanase stably overexpressing clones, wild-type (Control) and vector control (pcDNA3.1) PC12
cells treated with ( + ) or without ( − ) the p38 MAPK inhibitor SB203580 (20 μM). (D) Heparanase stably overexpressing cell clones, wild-type (Con) and vector control (pcDNA3.1) cells were
seeded on to 24-well plates at a density of 2 × 103 cells/well and cultured overnight, followed by treatment with medium (0.5 % horse serum) containing 25 ng/ml NGF in the presence or absence
of SB203580 (20 μM) for 72 h. The histogram shows statistical analysis of the proportion of cells bearing at least one neurite twice as long as the diameter of the cell body in human heparanase
stably overexpressing cell clones, wild-type (Con) and vector control (pcDNA3.1) cells induced by NGF (25 ng/ml) treated with or without SB203580 (20 μM). Data were from at least triplicate
experiments and are means +
− S.D. *P < 0.05, **P < 0.01 by Student’s t test. (E) Neurite outgrowth observation and images in (D) were taken as described in Figure 2. (F) Neuron differentiation
markers β-tubulin III and GAP-43 were measured by RT–PCR in heparanase stable silenced or overexpressing stable cell clones induce by NGF (25 ng/ml) compared with wild-type (Control) and
vector control cells (GFP or pcDNA3.1). 18S rRNA was used as an internal control.
enzymatic and non-enzymatic form. Since more EGR1, a
transcription factor of heparanase, is indeed recruited into the
nucleus by NGF (Figure 1D and Supplementary Figure S1) for
heparanase transcription, heparanase could be a component of
the TrkA-EGR1 pathway which modulates NGF-induced PC12
neuritogenesis.
Previous studies have demonstrated that sustained activation of
the ERK MAPK pathway is crucial for neuronal neuritogenesis
of PC12 cells [48,49]. However, the p38 MAPK signalling
pathway was also implicated in NGF-induced neuritogenesis
of PC12 cells [50]. NGF induces sustained activation of the
p38 MAPK signalling pathway in PC12 cells, and selective
blockade of this cascade results in inhibition of neurite outgrowth
[50]. Interestingly, neurite outgrowth induced by constitutively
activated MEK (MAPK/ERK kinase), considered a regulator of
c The Authors Journal compilation c 2011 Biochemical Society
ERK activity, also depends partly on p38 MAPK activity, although
MEK activates both ERK and p38 MAPKs. In fact, MEKinduced PC12 cell neurite outgrowth is sensitive to inhibition
of p38 MAPK by SB203580 [50]. Therefore a well-balanced
activation of the ERK and p38 MAPK pathways may be necessary
for neuritogenesis of PC12 cells in response to NGF. In the
present study, we found that overexpression of heparanase (both
wild-type and mutant latent heparanase molecules) promoted the
constitutive phosphorylation of p38 MAPK, whereas silencing
of heparanase attenuated the signal. However, phosphorylation
of ERK and JNK was not obviously changed in cells with
heparanase (wild-type and mutant) overexpressed or silenced.
Meanwhile, SB203580, an inhibitor of p38 MAPK, nearly
completely abolished the promotion of heparanase (wild-type and
mutant) on PC12 neuritogenesis. On the basis of these findings,
Heparanase augments neuritogenesis
Figure 5
281
Mutant heparanase enhanced NGF-induced neuritogenesis through p38 MAPK phosphorylation
(A) After the lentiviral vector was prepared for 48 h in HEK-293T cells, the latent heparanase (Hepa) mutant was infected into PC12 cells for 24 h and detected by Western blot analysis with an
anti-heparanase antibody. (B) Phospho-p38 MAPK (Thr180 /Tyr182 ), phospho-p44/42 MAPK (ERK1/2) and phospho-SAPK/JNK (Thr183 /Tyr185 ) were detected by Western blot analysis in mutant
human heparanase-overexpressing cells (ZS-M-HPSE), compared with wild-type (Control) and vector control cells (pLVX-ZS). Total p38 MAPK, p44/42 MAPK (ERK1/2) and β-actin proteins served
as loading controls. (C) The histogram shows the proportion of cells bearing at least one neurite twice as long as the diameter of the cell body in ZS-M-HPSE, control (Con) and pLVX-ZS cell
groups in the absence or presence of NGF (25 ng/ml). Morphology evaluation was performed as described in Figure 2. Detailed morphology images are shown in Supplementary Figure S5 (at
http://www.BiochemJ.org/bj/440/bj4400273add.htm).
we deduced that both wild-type and mutant heparanase enhanced
phosphorylation of p38 MAPK in PC12 cells, which promoted
NGF-induced PC12 cell neuritogenesis.
In conclusion, the present study demonstrated that heparanase
was involved in differentiation of PC12 cells via p38 MAPK
phosphorylation, whereas the latent form of heparanase might
play an independent role in neuritogenesis. Further studies are
necessary to determine whether this is a neural-restricted or a
more general mechanism in order to extend its relevance to adult
stem/multipotent cell differentiation.
AUTHOR CONTRIBUTION
Hengxiang Cui and Kan Ding designed this project; Hengxiang Cui, Chenghao Shao,
Qin Liu, Wenjie Yu, Jianping Fang, Weishi Yu and Amjad Ali performed experiments;
Hengxiang Cui and Chenghao Shao analysed data; Weishi Yu carried out the BrdU
analysis; Hengxiang Cui and Kan Ding wrote the paper.
FUNDING
The work was supported by the National Natural Science Foundation of China [grant
number 30770484]; and the National Science and Technology Major Project “Key New Drug
Creation and Manufacturing Program” [grant numbers 2009ZX09301-001, 2009ZX09501011, 2009ZX09103-071].
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Biochem. J. (2011) 440, 273–282 (Printed in Great Britain)
doi:10.1042/BJ20110167
SUPPLEMENTARY ONLINE DATA
Heparanase enhances nerve-growth-factor-induced PC12 cell
neuritogenesis via the p38 MAPK pathway
Hengxiang CUI*1 , Chenghao SHAO†1 , Qin LIU*, Wenjie YU‡, Jianping FANG*, Weishi YU‡, Amjad ALI‡ and Kan DING*2
*Glycochemistry and Glycobiology Laboratory, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China, †Department of Surgery, Changhai
Hospital, Shanghai 200433, China, and ‡The Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
Figure S1
Effect of NGF on formation of a EGR-1–DNA complex
Nuclear extracts (NE) isolated from PC-12 cells exposed to NGF for 60 and 120 min
were subjected to EMSA using 32 P-labelled double-stranded oligonucleotides encoding the
EGR-1-binding element in the PNMT promoter (5 -CCTCCCGCCCCCGCGCGTCC-3 ) as
a probe as described previously [33]. Protein–DNA complexes after separation on 5 %
polyacrylamide gels were visualized by autoradiography.
1
2
These authors contributed equally to this work
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
H. Cui and others
Figure S2 Effect on morphology of heparanase knock down on PC12
neuritogenesis induced by NGF
Figure S3 Effect on morphology of heparanase overexpression on PC12
neuritogenesis induced by NGF
A neuritogenesis assay of heparanase stably silenced cell clones (Sh-2, Sh-7 and Sh-12)
compared with the wild-type counterpart (Control) and vector control pGCsi-GFP (GFP) was
performed. Cells (2 × 103 cells/well) were seeded on 24-well plates and incubated overnight,
followed by exposure to medium (0.5 % horse serum) containing 25 ng/ml NGF for 72 h. Neurite
outgrowth was observed with nine random images captured per well, using either an inverted
fluorescence microscope or a phase-contrast microscope. The images were captured by a digital
camera at ×200. All cells in each image were analysed, with over 200 cells assessed per cell
culture plate. The experiments were repeated at least three times.
Heparanase-overexpressing cells (9406, 9415 and 9416), wild-type (Control) and sham cells
(pcDNA3.1) at a density of 2 × 103 cells/well were seeded and treated with NGF under the same
conditions as described in Figure S2. The images were taken as described in Figure S2. The
experiments were repeated at least three times.
c The Authors Journal compilation c 2011 Biochemical Society
Heparanase augments neuritogenesis
Figure S4 Effect of heparanase overexpression or knockdown on signalling of p38 MAPK, p44/42 MAPK (ERK1/2), SAPK/JNK and mRNA expression of tubulin
III and GAP43
Phospho-p44/42 MAPK (ERK1/2) (Thr202 /Tyr204 ) and phospho-SAPK/JNK (Thr183 /Tyr185 ) were detected by Western blot analysis in human heparanase (A) overexpressing (9406, 9415 and 9416)
and (B) stably silenced (Sh-2, Sh-7, Sh-12) PC12 cell clones compared with wild-type (Control in A and B) and vector control cells (pcDNA3.1 in A; GFP in B) respectively. Total p44/42 MAPK
(ERK1/2), SAPK/JNK and β-actin proteins were used as loading controls. (C and D) Phosphorylation of p38 MAPK normalized to total p38 MAPK in (A) and (B) are summarized. Con, wild-type
cells; pcDNA and GFP are vector control cells as described in (A) and (B). HHO, human heparanase-overexpressing cells (9406, 9415 and 9416); shRNA, heparanase stably silenced cells (Sh-2,
Sh-7, Sh-12). (E) Phospho-p44/42 MAPK (ERK1/2) (Thr202 /Tyr204 ) and phospho-SAPK/JNK (Thr183 /Tyr185 ) were detected in wild-type (Control), vector control (pcDNA3.1) and three heparanase
stably transfected cells (9406, 9415 and 9416). (F) Statistical summary of relative mRNA expression of tubulin III and GAP43 in heparanase stably silenced or heparanase-overexpressing stable cell
clones.
c The Authors Journal compilation c 2011 Biochemical Society
H. Cui and others
Figure S5 PC12 cells were treated with or without SB203580 and NGF for 72 h followed by analysis of the morphology in response to NGF with ( + ) or
without SB203580 (20 μM) in wild-type cells (Control), vector-control (pLVX-IRES-ZsGreen1) virus-infected cells (ZS) and mutant heparanase (pLVX-IRESZsGreen1-M-HPSE) virus-infected cells (M-HPSE)
The neuritogenesis assay was conducted as described in Figure S2. − NGF, without NGF; + NGF, with NGF (25 ng/ml).
Received 25 January 2011/9 August 2011; accepted 11 August 2011
Published as BJ Immediate Publication 11 August 2011, doi:10.1042/BJ20110167
c The Authors Journal compilation c 2011 Biochemical Society