IPP5 inhibits neurite growth in primary sensory neurons by ...€¦ · Accepted 14 November 2012 Journal of Cell Science 126, 542–553 2013. Published by The Company of Biologists

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IPP5 inhibits neurite growth in primary sensoryneurons by maintaining TGF-b/Smad signaling

Qing-Jian Han1, Nan-Nan Gao1, Guo-Qiang Ma1, Zhen-Ning Zhang1, Wen-Hui Yu1, Jing Pan1, Qiong Wang1,Xu Zhang2 and Lan Bao1,*1State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy ofSciences, Shanghai 200031, China2Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai 200031, China

*Author for correspondence ([email protected])

Accepted 14 November 2012Journal of Cell Science 126, 542–553� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.114280

SummaryDuring nerve regeneration, neurite growth is regulated by both intrinsic molecules and extracellular factors. Here, we found thatinhibitor 5 of protein phosphatase 1 (IPP5), a newly identified inhibitory subunit of protein phosphatase 1 (PP1), inhibited neurite

growth in primary sensory neurons as an intrinsic regulator. IPP5 was highly expressed in the primary sensory neurons of rat dorsal rootganglion (DRG) and was downregulated after sciatic nerve axotomy. Knocking down IPP5 with specific shRNA increased the length ofthe longest neurite, the total neurite length and the number of neurite ends in cultured rat DRG neurons. Mutation of the PP1-dockingmotif K8IQF11 or the PP1-inhibiting motif at Thr34 eliminated the IPP5-induced inhibition of neurite growth. Furthermore, biochemical

experiments showed that IPP5 interacted with type I transforming growth factor-b receptor (TbRI) and PP1 and enhanced transforminggrowth factor-b (TGF-b)/Smad signaling in a PP1-dependent manner. Overexpressing IPP5 in DRG neurons aggravated TGF-b-inducedinhibition of neurite growth, which was abolished by blocking PP1 or IPP5 binding to PP1. Blockage of TGF-b signaling with the TbRI

inhibitor SB431542 or Smad2 shRNA attenuated the IPP5-induced inhibition of neurite growth. Thus, these data indicate that selectivelyexpressed IPP5 inhibits neurite growth by maintaining TGF-b signaling in primary sensory neurons.

Key words: IPP5, Neurite growth, PP1, TGF-b/Smad signaling, DRG neuron

IntroductionNeurite growth is a critical step for successful nerve regeneration

after injury. Extracellular factors surrounding the ends of injured

axons were previously considered the major reasons affecting

axonal growth (Filbin, 2003; Schwab, 2004; Yiu and He, 2006).

However, recent evidence indicates that the activation of intrinsic

molecules also play an important role (Abe and Cavalli, 2008;

Chen et al., 2007; Liu et al., 2011; Sun and He, 2010). Several

protein kinases, transcription factors and growth-associated

molecules have been shown to be intrinsic molecules that

positively regulate axon growth (Chen et al., 2007; Gao et al.,

2004; Lorber et al., 2009; MacGillavry et al., 2009; Moore et al.,

2009; Seijffers et al., 2007). Downregulation or low activity of

these positive molecules causes reduced axonal growth and

failure of nerve regeneration. However, intrinsic molecules that

negatively regulate axonal growth are also considered important

when the cell is unable to eliminate or even activate these

molecules following nerve injury (Abe and Cavalli, 2008; Liu

et al., 2011). The transforming growth factor-b (TGF-b)/Smad

signaling pathway inhibits axon growth (de la Torre-Ubieta and

Bonni, 2011). Knocking down Smad2 or blocking the TGF-b/

Smad signaling pathway with SB431542 or specific antibodies

enables axons to override myelin inhibition and promotes

functional recovery after spinal cord injury (He and Wang,

2006; Kohta et al., 2009; Stegmuller et al., 2008).

Protein phosphatase 1 (PP1) is a eukaryotic protein serine/

threonine phosphatase that is important in regulating neuronal

morphology. Inactivation of PP1 by phosphorylation at Thr320 or

with a specific inhibitor, calyculin A, dramatically affects nerve

growth factor-induced neurite growth in PC12 cells (Li et al.,

2007; Reber and Bouron, 1995). In the central nervous system, F-

actin-associated PP1 promotes spine development and axonal

growth (Bielas et al., 2007; Oliver et al., 2002). Generally, each

PP1 holoenzyme is composed of a catalytic subunit and a

regulatory subunit. The regulatory subunit determines the

substrate specificity, subcellular localization, and diverse

cellular functions (Bollen et al., 2010). Previous studies report

that the TGF-b/Smad signaling pathway can be specifically

regulated by PP1. Under the control of the regulatory subunit

GADD34, PP1 is targeted to the type I transforming growth

factor-b receptor (TbRI) and inactivates this Ser/Thr kinase

receptor through dephosphorylating the juxta-membrane region

(Shi et al., 2004).

IPP5, a newly identified regulatory subunit of PP1, belongs to

the protein phosphatase 1 regulatory subunit 1 (PPP1R1) family,

which has a highly conserved PP1-docking motif and a PP1-

inhibiting motif. IPP5 inhibits the enzymatic activity of PP1 after

being phosphorylated within its PP1-inhibiting motif at Thr34

(Wang et al., 2008). IPP5 promotes tumor cell cycle progression

by accelerating the G1-S transition in a PP1-dependent manner

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(Wang et al., 2008), inhibits anchorage-dependent growth and

induces apoptosis of HeLa cells (Zeng et al., 2009). However, the

function of IPP5 in the nervous system has not been investigated.

In the present study, IPP5 was shown to be selectively expressed

in the dorsal root ganglion (DRG) neurons of rats. The DRG

contains pseudounipolar sensory neurons that extend their

peripheral terminals to peripheral tissues and their central

terminals to the dorsal horn of the spinal cord to convey

somatic sensory signals. Generally, DRG neurons cultured in

vitro generate multiple neurites, which reflects the growth ability

of these sensory neurons. We found that IPP5 inhibits neurite

growth in cultured DRG neurons by inhibiting PP1 activity and

maintaining TGF-b/Smad signaling. Blocking TGF-b/Smad

signaling and PP1 function impaired the IPP5-induced

inhibition of neurite growth. Thus, IPP5 is a novel intrinsic

molecule that negatively regulates neurite growth in primary

sensory neurons.

ResultsIPP5 is selectively expressed in primary sensory neurons

and downregulated after sciatic nerve axotomy

We searched the Unigene library of the National Center for

Biotechnology Information (NCBI) to identify genes that are

highly expressed in the DRG but are expressed at relatively low

levels in other tissues. In this process, we identified IPP5. By

applying the reverse transcription polymerase chain reaction

(PCR), we further confirmed that IPP5 was highly expressed in

the DRG of rats but showed no detectable expression in other

regions of the central nervous system, including the cortex,

cerebellum, hypothalamus, hippocampus, medulla oblongata,

pituitary gland and spinal cord (Fig. 1A). Amino acid sequence

alignment showed that IPP5 belonged to the PPP1R1 family,

possessing a highly conserved PP1-docking motif K8IQF11 and a

PP1-inhibiting motif R31RPTPA36 (Fig. 1B). We also analyzed

the expression patterns of the other two members of the PPP1R1

Fig. 1. The expression pattern of IPP5 in primary sensory neurons and spinal cord. (A) Distribution of IPP5, inhibitor-1 and DARPP-32 in rat nervous

system using reverse transcription PCR. (B) Alignment of amino acid sequences in the N-terminal region of IPP5, inhibitor-1 and DARPP-32 from human (h), rat

(r) and mouse (m). Residues shaded in yellow are 100% identical. The PP1-docking motif and inhibiting motif are indicated with black lines. (C) Representative

images and quantitative analysis of the coexpression of IPP5 (green) with nonpeptidergic small neuron marker IB4 binding or peptidergic small neuron marker

CGRP or myelinated neuron marker NF160/200 (all red) in DRG neurons. Scale bars: 50 mm. (D) Representative images of the coexpression of IPP5 (green) with

IB4 or CGRP (both red) in laminae I–V of the spinal cord. Spinal cord lamination is indicated with dashed lines. The dorsal–ventral (D–V) axis is indicated with a

double-headed arrow. Scale bars: 250 mm. (E) Relative mRNA expression of IPP5 in the DRG 2, 7, 14 and 28 days after sciatic nerve axotomy using real-time

PCR. (F) Representative immunoblotting (the upper panel) and quantitative data (the lower panel) show the protein level of IPP5 in the DRG 2, 7, 14 and 28 days

after sciatic nerve axotomy. The data were normalized to the control and presented as means 6 s.e.m. (n53). *P,0.05, **P,0.01 and ***P,0.001 versus

control.

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family, inhibitor-1, and dopamine and cAMP-regulated

phosphoprotein Mr 32 kDa (DARPP-32), which exhibited

universal distributions in nervous system but low expression in

the DRG of rats (Fig. 1A).

To further explore the cell-type characterization of IPP5 in

the DRG, we produced a rabbit polyclonal antibody against IPP5

and confirmed its specificity with antigen preabsorption

(supplementary material Fig. S1A). Immunohistochemistry

showed that 54.462.5% of the DRG neurons were IPP5

positive (n53856 out of 7414 counted neurons). We then

assessed IPP5 expression in two major subsets of small DRG

neurons with unmyelinated C-fibers or myelinated Ad-fibers,

which are known as isolectin B4 (IB4)-positive nonpeptidergic

neurons and calcitonin gene-related peptide (CGRP)-positive

peptidergic neurons, respectively. Double immunofluorescence

staining showed that 55.962.1% of IPP5-positive neurons were

IB4 positive (n52820 out of 5073 counted neurons) and

25.761.2% were CGRP positive (n51143 out of 4397 counted

neurons; Fig. 1C). Of these, 49.361.7% of the IPP5-positive

neurons (n51664 out of 3358 counted neurons) were labeled

with P2X3 receptor, a purinergic receptor that is selectively

expressed in nonpeptidergic small DRG neurons (supplementary

material Fig. S1C,D). Additionally, 14.460.9% of IPP5-positive

neurons (n5606 out of 4179 counted neurons) contained

Fig. 2. Knocking down IPP5 promotes neurite growth in cultured primary sensory neurons. (A) Schematic of IPP5 shRNA and untargeted shRNA-resistant

IPP5 (IPP5R) which was refractory to silencing by IPP5 shRNA. The red bracket above the amino acid sequence of IPP5 shows the targeting region of IPP5 shRNA,

and the nucleotides in red are the same sense mutation on IPP5. (B) Cultured DRG neurons were transfected with scrambled (SCB) shRNA, IPP5 shRNA or IPP5

shRNA and IPP5R, and subjected to immunoblotting using the indicated antibodies. The experiment was repeated at least three times. (C) ND7-23 cells were

transfected with plasmids coexpressing IPP5 or IPP5R with SCB shRNA or IPP5 shRNA, and subjected to immunoblotting using the antibodies indicated. The

experiment was repeated at least three times. (D) Representative DRG neurons expressing SCB shRNA, IPP5 shRNA or coexpressing IPP5 shRNA with IPP5R. Scale

bar: 200 mm. (E–G) Quantitative data show the effect of knocking down IPP5 on the length of the longest neurite (E), the total neurite length (F) and the number of

neurite ends (G) in cultured DRG neurons. The data were pooled from three independent experiments and normalized to control (SCB shRNA). The numbers in the

columns are the total number of neurons for each group. Data are means 6 s.e.m. (n53). *P,0.05, **P,0.01 and ***P,0.001 versus neurons expressing SCB

shRNA or as indicated. The cumulative frequency refers to the proportion of the total events. For the length of the longest neurite, P50.001 for IPP5 shRNA and

P.0.05 for IPP5 shRNA/IPP5R versus SCB shRNA, and P,0.001 for IPP5 shRNA/IPP5R versus IPP5 shRNA; for total neurite length, P,0.001 for IPP5 shRNA and

P.0.05 for IPP5 shRNA/IPP5R versus SCB shRNA, and P,0.001 for IPP5 shRNA/IPP5R versus IPP5 shRNA; for the number of neurite ends, P,0.05 for IPP5

shRNA and P.0.05 for IPP5 shRNA/IPP5R versus SCB shRNA, and P50.001 for IPP5 shRNA/IPP5R versus IPP5 shRNA (KS-test).

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neurofilament 160/200 (NF160/200), which labels large DRG

neurons (diameter .35 mm) with myelinated Ab-fibers

(Fig. 1C). Consistently, double immunofluorescence staining in

the dorsal horn of the spinal cord showed abundant IPP5-positive

nerve fibers in laminae I–II and a few IPP5-positive nerve fibers

in laminae III–IV (Fig. 1D). Most IPP5-positive nerve fibers

were located in the inner laminae II, an area that exhibited IB4

binding (Fig. 1D). Thus, IPP5 is predominantly expressed in

small primary sensory neurons that mainly project to laminae I–II

of the spinal cord.

We performed both real-time PCR and immunoblotting to

analyze the expression of IPP5 in L4 and L5 DRGs of rats after

sciatic nerve axotomy. The quantitative data from real-time PCR

showed that the mRNA level of IPP5 in the DRGs was reduced to

approximately 60% at post-nerve injury day 2 and maintained at

the same low levels until day 28 (Fig. 1E). Immunoblotting further

revealed that the reduction of IPP5 mRNA in the DRGs was

accompanied by decreased protein levels of IPP5. The

immunoblotting band with a molecular weight of approximately

16 kD was confirmed to be IPP5 by antigen preabsorption

(supplementary material Fig. S1B). The protein levels in the

DRGs were reduced to approximately 20% at post-nerve injury

day 7 and gradually recovered to approximately 60% at post-nerve

injury day 14 (Fig. 1F). Thus, IPP5 is remarkably downregulated

in DRG neurons after nerve injury and may serve as a neurite

growth inhibitory molecule in primary sensory neurons.

Knocking down IPP5 promotes neurite growth in cultured

primary sensory neurons

To examine the effect of IPP5 on neurite growth, we used a

plasmid-based method of IPP5 shRNA (Fig. 2A) to knock down

endogenous IPP5 in rat primary sensory neurons. We

electroporated scrambled or IPP5 shRNA plasmids into

dissociated DRG neurons. The IPP5 protein levels were

significantly decreased two days after transfection with IPP5

shRNA plasmids (Fig. 2B), indicating that the construct

efficiently interfered with endogenous IPP5 expression. We

also constructed a shRNA-resistant (R) IPP5 (IPP5R, Fig. 2A)

that was not silenced by IPP5 shRNA. The expression level of

IPP5 was unchanged after coexpressing the IPP5 shRNA plasmid

with IPP5R in ND7-23 cells (Fig. 2C), indicating that IPP5R is

not targeted by IPP5 shRNA. In cultured DRG neurons, the effect

of IPP5 shRNA on expression of endogenous IPP5 could be

rescued by IPP5R as well (Fig. 2B).

Importantly, two days after knocking down IPP5 in cultured

DRG neurons, the length of the longest neurite was increased by

Fig. 3. The interaction of IPP5 with PP1 is

necessary for its inhibition of neurite

growth in cultured primary sensory

neurons. (A) Schematic of the amino acid

sequences of IPP5 and its mutants. The amino

acids in red are the mutations.

(B) Protein of DRGs was immunoprecipitated

with IPP5-specific antibody and subjected to

immunoblotting with the indicated antibodies.

The experiment was repeated three times.

(C) Co-IP experiments with IPP5-specific

antibody to test the interaction of IPP5 or

IPP5-M with endogenous PP1 in HEK293T

cells expressing IPP5 or IPP5-M.

Representative images are shown from three

independent experiments. (D) Representative

images of DRG neurons overexpressing

control vector or IPP5 or IPP5-M. Scale bar:

200 mm. (E–G) Quantitative data showing the

effect of IPP5 interaction with PP1 on the

length of the longest neurite (E), the total

neurite length (F), and the number of neurite

ends (G) in cultured DRG neurons. The data

were pooled from three independent

experiments and normalized to control. The

numbers in the columns are the total number

of neurons for each group. Data are means 6

s.e.m. (n53). **P,0.01 and ***P,0.001

versus control neurons or as indicated. The

cumulative frequency refers to the proportion

of the total events. For the length of the

longest neurite, P,0.001 for IPP5 and

P.0.05 for IPP5-M versus control; for the

total neurite length, P,0.01 for IPP5 and

P.0.05 for IPP5-M versus control; for the

number of neurite ends, P50.001 for IPP5

and P.0.05 for IPP5-M versus control

(KS-test).

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approximately 24% (Fig. 2D,E), the total neurite length wasincreased by approximately 35% (Fig. 2D,F), and the number of

neurite ends was increased by approximately 18% (Fig. 2D,G).The cumulative frequency curves of all measures weresignificantly right-shifted in DRG neurons transfected withIPP5 shRNA (Fig. 2E–G). Coexpression of untargeted IPP5R

corrected the length of the longest neurite, the total neurite lengthand the number of neurite ends, which were increased by IPP5shRNA (Fig. 2D–G), indicating that the phenotype caused by

silencing IPP5 is not due to off-target effects. These resultssuggest that IPP5 is a negative regulator of neurite growth inprimary sensory neurons.

The interaction with and inhibition of PP1 is responsiblefor the effect of IPP5 on neurite growth in cultured primarysensory neurons

IPP5 has been identified as a novel inhibitory subunit of PP1 thatcontains both the PP1-docking motif and the PP1-inhibiting motif(Wang et al., 2008). The PP1-docking motif K8IQF11 is required

for IPP5 binding to PP1, and the threonine at position 34 (Thr34)

within the PP1-inhibiting motif is presumed to bind to the

active site of PP1 when phosphorylated (Svenningsson et al.,

2004; Wang et al., 2008). A co-immunoprecipitation (co-IP)

experiment confirmed that IPP5 interacts with PP1 in both rat

DRGs and HEK293T cells expressing IPP5 (Fig. 3B,C).

Consistent with the shRNA knockdown experiment, IPP5

overexpression in cultured rat DRG neurons (supplementary

material Fig. S2A,B) reduced the length of the longest neurite to

,63% of the control (Fig. 3D,E), reduced the total neurite length

to approximately 72% of the control (Fig. 3D,F), and reduced the

number of neurite ends to 84% of the control (Fig. 3D,G). In

addition, we also tested the effect of IPP5 on the percentage of

neurons bearing neurites. Overexpressing IPP5 for 24 hr in

cultured DRG neurons significantly decreased the percentage of

neurons bearing neurites, including IB4-binding and CGRP-

positive small DRG neurons and large DRG neurons with a

diameter .35 mm (supplementary material Fig. S3). To further

test whether the regulation of neurite growth by IPP5 depends on

its interaction with PP1, we produced an IPP5 mutant (IPP5-M)

in which the PP1-docking motif K8IQF11 was replaced with

Fig. 4. The inhibitory activity of IPP5 to PP1 is necessary for

its inhibition of neurite growth in cultured primary sensory

neurons. (A) In vitro phosphorylation of IPP5 by PKA. Equal

amount of purified IPP5 or IPP5T34D protein (200 ng) were

incubated with PKA in the presence of [c-32P]ATP, and

subjected to detection by autoradiography. (B) In vitro

phosphorylation of IPP5 by PKA with or without treatment with

the PKA inhibitor H89 was tested with site-specific monoclonal

antibody against phosphorylated IPP5 at Thr34. (C) The cultured

DRG neurons were harvested for immunoblotting 10 min after

10 mM forskolin treatment with or without 2 mM cyclosporin A

(CsA). (D) Representative images of DRG neurons

overexpressing control vector, IPP5, IPP5T34A or IPP5T34D. Scale

bar: 200 mm. (E–G) Quantitative data showing the effect of the

different status of IPP5 phosphorylation on the length of the

longest neurite (E), the total neurite length (F), and the number of

neurite ends (G) in cultured DRG neurons. The data were pooled

from three independent experiments and normalized to control.

The numbers in the columns are the total number of neurons for

each group. The data are means 6 s.e.m. (n53). *P,0.05,

**P,0.01 and ***P,0.001 versus control neurons or as

indicated. The cumulative frequency refers to the proportion of

the total events. For the length of the longest neurite, P,0.001

for IPP5 and IPP5T34D, and P.0.05 for IPP5T34A versus control;

for the total neurite length, P,0.001 for IPP5 and IPP5T34D, and

P.0.05 for IPP5T34A versus control; for the number of neurite

ends, P,0.001 for IPP5 and IPP5T34D, and P.0.05 for IPP5T34A

versus control (KS-test).

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alanine (Fig. 3A). Co-IP experiments showed that the interaction

between PP1 and IPP5-M was dramatically reduced compared

with that between PP1 and wild-type IPP5 in HEK293T cells

(Fig. 3C). Correspondingly, IPP5-M had no significantly

inhibitory effect on the length of the longest neurite, the total

neurite length or the number of neurite ends (Fig. 3D–G). These

data indicate that the interaction of IPP5 with PP1 is essential for

its inhibition of neurite growth.

Previous in vitro protein phosphatase assays have shown that

IPP5 phosphorylated at Thr34 and IPP5T34D (Fig. 3A), a

phosphorylation-mimicking mutant, were able to effectively

inhibit PP1 activity with IC50 values of 45 nM and 110 nM,

respectively, while IPP5T34A (Fig. 3A), a phosphorylation-

deficient mutant, had no effect on PP1 (Wang et al., 2008).

Our in vitro autoradiograph assay showed that IPP5 was

phosphorylated by PKA but IPP5T34D was not (Fig. 4A). The

phosphorylation of IPP5 was also detected by a site-specific

monoclonal antibody against phosphorylated Thr34, which was

blocked by the PKA pharmacological inhibitor H89 (Fig. 4B).

Further pharmacological assays showed that the phosphorylation

level of IPP5 at Thr34 was dramatically elevated by treating

cultured DRG neurons with the cAMP/PKA signaling pathway

activator forskolin and was further enhanced by the protein

phosphatase 2B (PP2B) inhibitor cyclosporin A (Fig. 4C). Thus,

Thr34 within the PP1-inhibiting motif of IPP5 is phosphorylated

by PKA and dephosphorylated by PP2B, similar to the other

PPP1R1 family members, inhibitor-1 and DARPP-32 (Endo et al.,

1996; Nairn et al., 2004; Weiser et al., 2004).

To further investigate whether the regulation of neurite growth

by IPP5 depends on its inhibition against PP1, dissociated DRG

neurons were electroporated with IPP5 or its two phosphorylation

mutants, IPP5T34A and IPP5T34D. Similar to the cultured DRG

neurons expressing IPP5, the length of the longest neurite, the

total neurite length and the number of neurite ends were

markedly decreased in neurons expressing IPP5T34D but not

IPP5T34A (Fig. 4D–G). Taken together, these data indicate that

both the interaction with and inhibition of PP1 are indispensable

for the negative regulation of neurite growth by IPP5.

IPP5 maintains TGF-b signaling

Given that PP1 mediates the effect of IPP5 on neurite growth in

primary sensory neurons, we searched for the downstream

pathway. Several signaling molecules have been reported to be

both regulated by PP1 and involved in the regulation of neurite

growth (Abe et al., 2010; Bito et al., 1996; Gao et al., 2004;

Hur et al., 2011; Morfini et al., 2004; Shi et al., 2004; Stegmuller

et al., 2008; Thayyullathil et al., 2011; Xiao et al., 2010; Zhou

et al., 2004). Dephosphorylation of Akt/glycogen synthase kinase

3 beta (GSK3b), cAMP-response element binding protein

(CREB) and TbRI by PP1 regulates the phosphatidylinositol

3-kinase (PI3K), cAMP/PKA and TGF-b/Smad signaling

pathways, respectively. To ascertain whether these signaling

molecules are regulated by IPP5, we expressed IPP5 or its

phosphorylation mutants, IPP5T34A and IPP5T34D, in HEK293T

cells. The basal phosphorylation levels of CREB, Akt and

GSK3b were high and unchanged in cells expressing IPP5 or its

mutants (Fig. 5A). However, the basal phosphorylation levels of

Smad2 and Smad3, two molecules downstream of TbRI, were

almost undetectable in HEK293T cells. We treated cells with

10 ng/ml TGF-b1 for 60 min to increase the phosphorylation

level of Smad2/3 and found that Smad2/3 phosphorylation was

upregulated in cells expressing IPP5 and IPP5T34D compared to

cells expressing the control vector (Fig. 5B). Importantly, the

phosphorylation level of Smad2/3 was not affected in cells

expressing IPP5-M or IPP5T34A (Fig. 5B). This result suggests

that IPP5 positively regulates the TGF-b/Smad signal pathway,

and the interaction with and inhibition of PP1 are also essential

for IPP5-mediated Smad2/3 activation.

Fig. 5. IPP5 regulates TGF-b/Smad signaling.

(A) HEK293T cells were transfected with plasmids

expressing IPP5, IPP5T34A or IPP5T34D, and subjected

to immunoblotting with the indicated antibodies.

(B) HEK293T cells expressing IPP5, IPP5T34A,

IPP5T34D or IPP5-M were treated with vehicle or

10 ng/ml TGF-b1 for 60 min and subjected to

immunoblotting with the indicated antibodies. (C) The

dissociated DRG neurons were fixed for

immunocytochemistry with the indicated antibodies.

Scale bars: 15 mm. (D) Cultured DRG neurons were

transfected with control siRNA or IPP5 siRNA, treated

with vehicle or 10 ng/ml TGF-b1 for 60 min and then

subjected to immunoblotting with the indicated

antibodies. (E) Cultured DRG neurons were left

untreated or treated with 10 ng/ml TGF-b1 for 60 min,

harvested and immunoprecipitated with IPP5-specific

antibody, and immunoblotted with the indicated

antibodies. (F) HEK293T cells were transfected with

IPP5 and HA-TbRI, harvested and

immunoprecipitated with an antibody against HA, and

immunoblotted with the indicated antibodies. All

experiments were repeated at least three times.

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We also tested whether IPP5 regulates the TGF-b signaling

pathway in primary sensory neurons. Immunocytochemistry

showed that IPP5 colocalized with PP1 and TbRI in the region

near the plasma membrane in dissociated DRG neurons

(Fig. 5C). After TGF-b1 stimulation, the distributions of IPP5,

PP1 and TbRI were not obviously changed, but the

phosphorylation level of Smad2/3 within the nucleus was

significantly increased (supplementary material Fig. S4A,B).

IPP5 siRNA was employed to efficiently knock down

endogenous IPP5 in order to perform biochemical assays in

cultured DRG neurons. The phosphorylation level of Smad2/3

after treatment with 10 ng/ml TGF-b1 for 60 min was reduced in

cultured DRG neurons transfected with IPP5 siRNA compared to

control siRNA (Fig. 5D). These data suggest that IPP5 regulates

the TGF-b/Smad signaling pathway in primary sensory neurons.

Signaling by TGF-b family members mainly occurs via type I

and type II serine/threonine kinase receptors. Binding of TGF-b1

dimers to TbRII leads to the recruitment of TbRI and the formation

of a tetrameric complex. Constitutively active TbRII activates

TbRI by specifically phosphorylating its serine and threonine

residues in the juxta-membrane region. Activated TbRI propagates

the signal downstream by directly phosphorylating Smad2 and

Smad3 (ten Dijke and Hill, 2004). Previous studies have reported

that PP1 interacts with TbRI and inhibits its function via

dephosphorylation (Shi et al., 2004). Our co-IP experiments

showed that IPP5 interacts with both TbRI and PP1 in cultured

DRG neurons. This interaction was not enhanced by TGF-btreatment (Fig. 5E). Reverse-IPs in HEK293T cells transiently

expressing IPP5 and HA-TbRI also showed an interaction of TbRI

with IPP5 and PP1 (Fig. 5F). Thus, IPP5 forms a protein complex

Fig. 6. IPP5 enhances the TGF-b-induced inhibition of neurite growth in a PP1-dependent manner. (A) Representative images of DRG neurons

overexpressing control vector, IPP5 or IPP5-M treated with vehicle or 10 ng/ml TGF-b1 together with or without 5 nM tautomycin (Tau). Scale bar: 100 mm.

(B–D) Quantitative data showing the effect of IPP5 on the TGF-b-induced inhibition of neurite growth, including the length of the longest neurite (B), the total

neurite length (C) and the number of neurite ends (D). The data were pooled from three independent experiments and normalized to the control. The numbers in

the columns are the total number of neurons for each group. The data are means 6 s.e.m. (n53). *P,0.05, **P,0.01 and ***P,0.001 versus control neurons or

as indicated. The cumulative frequency refers to the proportion of the total events. For the length of the longest neurite, P,0.001 for TGF-b1, TGF-b1/IPP5, TGF-

b1/Tau, TGF-b1/Tau/IPP5, and TGF-b1/IPP5-M versus control, and P,0.05 for TGF-b1/IPP5 and versus TGF-b1, and P,0.001 for TGF-b1/IPP5-M versus

TGF-b1/IPP5, P.0.05 for TGF-b1/tau versus TGF-b1/tau/IPP5; for the total neurite length, P,0.001 for TGF-b1, TGF-b1/IPP5, TGF-b1/Tau, TGF-b1/Tau/

IPP5, and TGF-b1/IPP5-M versus control, and P,0.001 for TGF-b1/IPP5 versus TGF-b1, P.0.05 for TGF-b1/IPP5-M versus TGF-b1, and TGF-b1/tau versus

TGF-b1/tau/IPP5; for the number of neurite ends, P,0.01 for TGF-b1, TGF-b1/IPP5, TGF-b1/Tau, TGF-b1/Tau/IPP5, and TGF-b1/IPP5-M versus control, and

P.0.05 for TGF-b1/IPP5, and TGF-b1/IPP5-M versus TGF-b1, P.0.05 for TGF-b1/tau versus TGF-b1/tau/IPP5 (KS-test).

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with TbRI and PP1, which may maintain TGF-b/Smad signalingby inhibiting the dephosphorylation of TbRI by PP1.

IPP5 inhibits neurite growth by regulating the TGF-bsignaling pathway

The TGF-b/Smad signaling pathway has been reported to be anintrinsic negative regulator of neurite growth (He and Wang,2006; Hellal et al., 2011; Kohta et al., 2009; Ng, 2008;

Stegmuller et al., 2008). Considering the effect of IPP5 on TGF-b/Smad signaling, we investigated the involvement of the TGF-b/Smad pathway in the IPP5-induced inhibition of neurite

growth. Reverse transcription PCR showed that TGF-b1, TGF-b2, and TGF-b3 were expressed in both DRGs and culturedDRG neurons (supplementary material Fig. S4C). We then

performed neurite growth assays in the presence of TGF-bs. Thedata showed that the length of the longest neurite, the totalneurite length and the number of neurite ends were dramatically

inhibited by TGF-bs (Fig. 6; supplementary material Fig. S4D–G). TGF-b1-induced inhibition of the length of the longestneurite and the total neurite length were further enhanced byIPP5 overexpression, but this effect was not observed with

regard to the number of neurite ends (Fig. 6). Treatment with5 nM tautomycin, a PP1 inhibitor, prevented the enhancementof the TGF-b1-induced neurite growth inhibition by IPP5

(Fig. 6). Similarly, IPP5-M, which lacks the ability to bind PP1,did not enhance the TGF-b1-induced inhibition of neuritegrowth (Fig. 6). These data show that IPP5 enhances the TGF-b-

induced inhibition of neurite growth in a PP1-dependentmanner.

A drug (SB-431542) has been reported to be a potent and

specific inhibitor of the TGF-b receptor superfamily. Itdramatically inhibits the kinase activity of TbRI, the activintype I receptor and the nodal type I receptor, but it has little

effect on other protein kinases (Inman et al., 2002). Consideringexpression of TGF-bs in cultured DRG neurons (supplementarymaterial Fig. S4C), activation of TGF-b signaling wasmaintained in basal condition (Fig. 5C). Inhibition of TGF-bsignaling with 5 mM SB431542 increased the length of thelongest neurite by approximately 27%, the total neurite lengthby approximately 37%, and the number of neurite ends by

approximately 12.7% in cultured rat DRG neurons (Fig. 7).Importantly, the IPP5-induced inhibition of neurite growth wasblocked by 5 mM SB431542 treatment (Fig. 7). To search for a

more specific way to block the activation of the TGF-b/Smadsignaling pathway, we selected a Smad2 shRNA to knock downendogenous Smad2 in DRG neurons (supplementary material

Fig. S5A). In cultured DRG neurons expressing Smad2 shRNA,the inhibition of the TGF-b signaling increased the length of thelongest neurite by approximately 23% and the total neuritelength by approximately 25% (Fig. 8A–C). Furthermore, the

IPP5-induced inhibition of neurite growth was blocked incultured DRG neurons coexpressing Smad2 shRNA (Fig. 8A–C). Taken together, these data indicate that the activation of the

TGF-b/Smad signaling pathway is necessary for the IPP5-induced inhibition of neurite growth in primary sensoryneurons.

DiscussionIPP5 is a PP1-inhibiting protein that has been shown to regulatetumor cell cycle, growth and apoptosis (Wang et al., 2008; Zenget al., 2009). Here, we report a critical role of IPP5 in the

inhibition of neurite growth in primary sensory neurons. IPP5

was selectively expressed in the DRG in the nervous system,

specifically in small primary sensory neurons. If the ability of

Fig. 7. Specific inhibition of TGF-b receptor eliminates the IPP5-induced

inhibition of neurite growth. (A) Representative images of DRG neurons

overexpressing control vector or IPP5 with vehicle or 5 mM SB431542 treatment.

Scale bar: 200 mm. (B–D) Quantitative data showing the effect of TGF-b

receptor inhibition with SB431542 on the IPP5-induced inhibition on the length

of the longest neurite (B), the total neurite length (C), and the number of neurite

ends (D) in cultured DRG neurons. The data were pooled from three independent

experiments and normalized to the control. The numbers in the columns are the

total number of neurons for each group. The data are means 6 s.e.m. (n53).

*P,0.05, **P,0.01 and ***P,0.001 versus control neurons or as indicated.

The cumulative frequency refers to the proportion of the total events. For the

length of the longest neurite, P,0.01 for IPP5 and control/SB431542 versus

Control, and P.0.05 for IPP5/SB431542 versus control/SB431542; for the total

neurite length, P50.001 for IPP5 and Control/SB431542 versus control, and

P.0.05 for IPP5/SB431542 versus control/SB431542; for the number of neurite

ends, P50.001 for IPP5 and control/SB431542 versus control, and P.0.05 for

IPP5/SB431542 versus control/SB431542 (KS-test).

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IPP5 to interact with or inhibit PP1 was disrupted, the inhibitory

effects of IPP5 on neurite growth were impaired. IPP5 formed a

protein complex with TbRI and PP1 and maintained TGF-b/

Smad signaling by inhibiting the function of PP1 (Fig. 8D).

Furthermore, IPP5 enhanced the TGF-b-induced inhibition of

neurite growth. Blockage of TGF-b/Smad signaling disrupted the

IPP5-induced inhibition of neurite growth. These findings extend

our understanding about the function of the regulatory subunits of

PP1 in the nervous system.

IPP5 inhibits neurite growth in primary sensory neurons ina PP1-dependent manner

Phosphorylation and dephosphorylation of cellular proteins by

protein kinases and phosphatases are important mechanisms for

controlling many cellular events. In the nervous system, protein

phosphatases are contained in highly dynamic complexes localized

within specialized subcellular compartments to ensure the

temporally and spatially controlled dephosphorylation of multiple

neuronal phosphoproteins (Mansuy and Shenolikar, 2006).

Approximately ten different regulatory subunits of PP1 have been

identified in the nervous system (Cohen, 2002). Neurabin-I and

spinophilin are both regulatory subunits targeting PP1 to F-actin

during neuronal development, which regulates spine development

or axonal growth by affecting the cytoskeletal dynamics in

hippocampal and cortical neurons (Bielas et al., 2007; Oliver

et al., 2002). Scapinin is another PP1-inhibiting protein that inhibits

axonal growth without affecting neurite branching in primary rat

cortical neurons (Farghaian et al., 2011). As a regulatory subunit of

PP1, IPP5 is selectively expressed in primary sensory neurons

localized in the DRG. Knocking down IPP5 significantly elevated

neurite growth in cultured DRG neurons, indicating that IPP5

functions as a negative regulator of neurite growth. This conclusion

is further supported by the evidence that IPP5 overexpression

significantly inhibited neurite growth in cultured DRG neurons.

IPP5 is the first regulatory subunit of PP1 identified in primary

sensory neurons that exhibits this function.

Previous studies reported that members of the PPP1R1 family

function by regulating PP1-dependent signaling pathways and

inhibiting PP1 activity (Bollen et al., 2010; Nairn et al., 2004).

Our study also suggests that IPP5 inhibits neurite growth in a

PP1-dependent manner. Being a member of the PPP1R1 family,

IPP5 possesses a highly conserved PP1-docking motif K8IQF11 in

the N-terminus and a PP1-inhibiting motif R31RPTPA36. Loss of

IPP5 binding or inhibitory activity regarding PP1 and blockage of

PP1 activity disrupted the IPP5-induced inhibition of neurite

growth in cultured DRG neurons. In contrast, increasing IPP5

inhibitory activity regarding PP1 by expressing IPP5T34D

inhibited neurite growth. However, the inhibitory effect of

IPP5T34D on neurite growth was slightly weaker than that of wild-

type IPP5, which is consistent with previous evidence showing

lower inhibitory activity of IPP5T34D towards PP1 compared to

wild-type IPP5 (Wang et al., 2008). The weaker inhibition of PP1

caused by IPP5T34D might be due to a conformational difference

from phosphorylated IPP5 at Thr34, which leads to a reduced

affinity for the PP1 catalytic subunit (Weiser et al., 2004).

Fig. 8. Knocking down Smad2 relieves the IPP5-induced inhibition of

neurite growth. (A) Representative images of DRG neurons coexpressing

control vector, IPP5 with control shRNA or Smad2 shRNA. Scale bar:

200 mm. (B,C) Quantitative data showing the inhibitory effect of TGF-b

signaling with Smad2 shRNA on the IPP5-induced inhibition of the length of

the longest neurite (B) and the total neurite length (C) in cultured DRG

neurons. The data were pooled from three independent experiment and

normalized to control. The numbers in the columns are the total number of

neurons for each group. The data are means 6 s.e.m. (n53). *P,0.05,

**P,0.01 and ***P,0.001 versus control neurons or as indicated. The

cumulative frequency refers to the proportion of the total events. For the

length of the longest neurite, P,0.01 for IPP5/control shRNA and control/

Smad2 shRNA versus control/control shRNA, and P.0.05 for IPP5/Smad2

shRNA versus control/Smad2 shRNA; for the total neurite length, P,0.001

for IPP5/control shRNA and control/Smad2 shRNA versus control/control

shRNA, and P.0.05 for IPP5/Smad2 shRNA versus control/Smad2 shRNA

(KS-test). (D) A model for IPP5 regulation of the TGF-b/Smad signaling

pathway and inhibition of neurite growth in primary sensory neurons. After

dimerizing TGF-b binds to TbRII, the TbRII recruits and activates TbRI by

phosphorylating its GS domain. PP1 could be targeted to the activated TbRI,

and inactivate TbRI by dephosphorylating its GS domain. IPP5 binds to PP1

and blocks PP1 activity, hence maintains the TGF-b/Smad signaling.

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The TGF-b/Smad signaling pathway mediates the

IPP5-induced inhibition of neurite growth

Regulatory subunits endow PP1 with distinct substrate

specificities and restricted subcellular locations (Cohen, 2002).

It is important to identify the phosphoprotein substrates of the

IPP5-PP1 holoenzyme complex. The phosphorylation levels of

CREB, Akt and GSK3b were not altered in cells expressing IPP5

and its mutants. Importantly, in HEK293T cells expressing IPP5

or IPP5T34D, the phosphorylation level of Smad2/3 was

significantly elevated after TGF-b stimulation. In cultured DRG

neurons, the TGF-b-induced phosphorylation of Smad2/3 was

reduced after knocking down IPP5. Thus, IPP5 positively

regulates TGF-b/Smad signaling. PP1 has been shown to

dephosphorylate activated TbRI but not Smad2/3, which is an

effective negative feedback mechanism for regulating TGF-b/

Smad signaling (Lin et al., 2006; Shi et al., 2004). IPP5, TbRI

and PP1 were found to colocalize in the region near the plasma

membrane in dissociated DRG neurons. Importantly, IPP5 was

shown to interact with TbRI and PP1, which formed a complex in

DRG neurons. In HEK293T cells expressing the IPP5 mutants

that lack PP1-docking or inhibiting activities, the

phosphorylation levels of Smad2/3 were not further elevated

after TGF-b stimulation. These data suggest a model in which

IPP5 inhibits PP1 activity, keeps TbRI activity, and maintains

TGF-b/Smad signaling (Fig. 8D).

During the development of the nervous system, TGF-b/Smad

signaling is critical for establishing neuronal polarity and axonal

identity (Awasaki et al., 2011; Farkas et al., 2003; Ng, 2008;

Stegmuller et al., 2008; Yi et al., 2010). Loss of the TGF-breceptor homolog Baboon resulted in neurite overextension in

neurons in drosophila mushroom body (Ng, 2008). Blockage of

the TGF-b signaling pathway with Taxol facilitated axonal

regeneration by decreasing scar formation and enhancing

intrinsic axonal growth (Hellal et al., 2011). In cultured cortical

neurons, TGF-b-induced inhibition of axonal growth was

eliminated by SB431542 treatment or Smad2 knock-down

(Stegmuller et al., 2008; Ylera et al., 2009). However, in RGC-

5 cells transformed form retinal ganglion cells TGF-b promoted

neurite growth through a noncanonical TGF-b/Smad signaling

pathway (Walshe et al., 2011). In our study, TGF-bs exhibited an

inhibitory effect on neurite growth and blocking TGF-b/Smad

signaling with the TbRI inhibitor SB431542 or Smad2 shRNA

also increased neurite growth, which support a role for TGF-bsignaling in the negative regulation of growth in DRG neurons.

Moreover, IPP5 enhanced the TGF-b-induced inhibition of

neurite growth and blocking TGF-b/Smad signaling with

SB431542 or Smad2 shRNA impaired the IPP5-induced

inhibition of neurite growth. Thus, the TGF-b/Smad signaling

pathway is essential to the function of IPP5 in primary sensory

neurons.

Functional implications of IPP5 in vivo

In this study, IPP5 was found to be highly expressed in the

peripheral nervous system but not in the central nervous system,

including the cortex, cerebellum, hypothalamus, hippocampus,

medulla oblongata, pituitary gland and spinal cord. Two other

members of the PPP1R1 family, inhibitor-1 and DARPP-32,

exhibited universal distributions in the nervous system but low

expression in the DRG. This expression pattern suggests that

IPP5 is a member of the PPP1R1 family that is expressed

specifically in primary sensory neurons to help PP1 recognize

specific substrates and restrict its subcellular locations.

Importantly, primary sensory neurons that expressed IPP5

projected to peripheral tissues and lamina I–II of the dorsal hornin the spinal cord. Axonal growth in the spinal cord is

dramatically inhibited after injury, even before the physicalobstacle of the glial scar is formed. This suggests that the spinal

cord provides an inhibitory environment for injured axons

because of the activation of several inhibitory signalingpathways. TGF-b is an important factor secreted by local

astrocytes at injury sites to stimulate the proliferation of

astrocytes and form a glial scar. TGF-b also inhibits the growthof injured axons (Kohta et al., 2009; Stegmuller et al., 2008). The

enrichment of IPP5 in the afferent fibers of primary sensoryneurons in the spinal cord might aggravate the inhibition of

axonal growth by maintaining activated TGF-b signaling. After

nerve injury, IPP5 in the DRG is remarkably downregulated,indicating the activation of a mechanism to increase the intrinsic

capacity for neurite growth in primary sensory neurons.

Interestingly, IPP5 is dominantly expressed in small primarysensory neurons. Other inhibitory subunits expressed in large

primary sensory neurons may also regulate PP1 function.

Materials and MethodsPlasmid construction

The expression construct of IPP5 was generated by inserting coding sequence ofthe PCR-amplified IPP5 (NM_001109200) from DRG cDNA of Sprague-Dawleyrat into pMyc vector (Clontech Laboratories, Palo Alto, CA) in which Myc tag wassubstituted for EGFP. The primers for amplifying coding sequence of IPP5 fromDRG were as follows: 59-tacaagcttatggagcccaac-39 and 59-tacggatccatggttccactt-39. Myc-IPP5T34, Myc-IPP5T34D, Myc-IPP5-M and Myc-1PP5R plasmids weregenerated by PCR from Myc-IPP5 using KOD-Plus-mutagenesis kit (Toyobo,Osaka, Japan) with following primers: 59-gatcaggaaaagaagacctgccccagcatccc-ttgtgattc-39 and 59-gaatcacaagggatgctggggcaggtcttcttttcctgatc-39; 59-gatcaggaaaaa-agacctgacccagcatcccttgtgattc-39 and 59-gaatcacaagggatgctgggtcaggtcttcttttcctgatc-39; 59-atggagcccaacagccccaaagcagctgcagctgctgtgcctttattccag-39 and 59-ctggaataaa-ggcacagcagctgcagctgctttggggctgttgggctccat-39; 59-cggaagaagaagaatcagcgtcggaga-gagaagaaaagtgg-39 and 59-ccacttttcttctctctccgacgctgattcttcttcttccg-39, respectively.The coding sequence of IPP5 and its mutants were also subcloned into pIRES-EGFP (Clontech Laboratories) or pCAG-IRES-EGFP, a modified vector ofpDC316 (VGTC, Beijing, China) in which the CMV promoter was replaced byCAG promoter. The scramble shRNA and IPP5 shRNA were cloned into pSuperRNAi vector (Oligoengine, Seattle, WA) with primers: 59-gatccccgagtgagaga-acacagagattcaagagatctctgtgttctctcactctttttggaaa-39 and 59-agcttttccaaaaagagtgag-agaacacagagatctcttgaatctcttgttctctcactcggg-39; 59-gatccccgcgcaagtgaaagagaagattc-aagagatcttctctttcactgcgctttttggaaa-39 and 59-agcttttccaaaaagcgcaagtgaaagagaaga-tctcttgaatcttctctttcacttgcgcggg-39. The control shRNA and Smad2 shRNA werecloned into U6 GFP RNAi vector (GenePharma, Shanghai, China) with primers:59-caccgttctccgaacgtgaacgtgtcacgtcaagagattacgtgacacgttcggagaatttttg-39 and 59-gatccaaaaaattctccgaacgtgtcacgtaatctcttgacgtgacacgttcggagaac-39; 59-caccgcgatcga-gaactgcgaatacttcaagagagtattcgcagttctcgatcgcttttttg-39 and 59-gatccaaaaagcgat-cgagaactgcgaatactctcttgaagtattcgcagttctcgatcgc-39. For the expression andpurification of GST-fused proteins, coding sequences of IPP5 and IPP5T34D

were subcloned into pGEX-4T1 vector (Amersham Pharmacia Biotech,Piscataway, NJ) with primers 59-gcggatccatggagcccaacagcccc-39 and 59-ccgaattcttaatggttccacttttcttc-39.

Animal model

Adult rats (body weight 250 g; Shanghai Center for Experimental Animals,Chinese Academy of Sciences, China) were used according to the policy of theSociety for Neuroscience (USA) regarding the use of animals. The experiment wasapproved by the Committee for the Use of Laboratory Animals and CommonFacilities, Institute of Biochemistry and Cell Biology, Chinese Academy ofSciences. For animal modeling of a peripheral nerve injury, a 5 mm portion of ratsciatic nerve was transected at mid-thigh level. The rats were killed after 2, 7, 14 or28 days (10 rats for each time point).

PCR

Total RNA was isolated from the tissues of adult rats using TRIzol reagent(Invitrogen, Carlsbad, CA). The first-strand cDNA was generated usingSuperScriptHII Reverse Transcriptase (Invitrogen) for reverse transcription PCR,

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and the products were analyzed on a 1% agarose gel. Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) was used as an internal control. The real-time PCR wasperformed in triplicate with SYBR Premix Ex Taq (TaKaRa, Shiga, Japan) on anABI 7500 real-time PCR system (Applied Biosystems, Carlsbad, CA), and theendogenous mRNA values of IPP5 were normalized to that of GAPDH. Theprimers were as follows: 59-cctacaccagcatcccttgt-39 and 59-ttgcgctttcttcttcttcc-39

for IPP5; 59-aagaacctgagggagccact-39 and 59-tgggaatccagtggtagcat-39 forInhibitor-1; 59-cacccaaagtcgaagagacc-39 and 59-tcatcctcgtcctcatcctc-39 forDARPP-32; and 59-ggcaagttcaacggcacag-39 and 59-cgccagtagactccacgac-39

for GAPDH.

Immunohistochemistry and immunocytochemistry

Adult rats were anesthetized and perfused through the ascending aorta with salinefollowed by 4% paraformaldehyde containing 0.02% picric acid. Lumbar (L)4 andL5 DRGs and the L4–L5 segment of the spinal cord were isolated, post-fixed, andcryoprotected in 20% sucrose. For both the DRG and the spinal cord, 14 mmsections were cut with a cryostat (Leica, Heidelberg, Germany). The sections wereincubated overnight with a rabbit antibody against IPP5 (1:5000; homemade)mixed with a mouse antibody against NF160/200 (1:10,000; Sigma, St Louis, MO)or a goat antibody against CGRP (1:1000; Biogenesis, Poole, UK) or a guinea pigantibody against P2X3 receptor (1:2000, Chemicon, Temecula, CA) or guinea pigantibody against substance P (SP, 1:1000; Neuromics, Minneapolis, MN). Then,the sections were incubated with secondary antibodies conjugated to FITC andCy3 (1:100; Jackson ImmunoResearch, West Grove, PA) and examined under aLeica TCS SP5 MP confocal microscope (Leica, German). To label the IB4-positive small DRG neurons, the sections were incubated with fluorescein-labeledIB4 (1:100; Vector Laboratories, Burlingame, CA). Two sections from each DRGwere quantitatively analyzed for each rat, and the data were collected from at leastthree animals. To determine the percentage of IPP5-positive neurons, the numberof stained neurons was divided by the total number of neurons. The percentage ofIPP5-positive neurons within a subset of primary sensory neurons was alsodetermined.

DRG neurons cultured on coverslips were fixed in 4% paraformaldehyde for15 min and incubated overnight with a rabbit antibody against IPP5 mixed with amouse antibody against PP1 (1:1000; Epitomics) or with a mouse antibody againstIPP5 (1:5000; Abcam, Cambridge, UK) mixed with a rabbit antibody against TbRI(1:500; Santa Cruz Biotechnology, Santa Cruz, CA) or phosphorylated Smad2/3(1:1000; Epitomics, Burlingame, CA) (supplementary material Fig. S5B) followedby secondary antibodies.

Cell culture and transfection

HEK293T cells (American Type Culture Collection, Manassas, VA) were culturedin MEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum(Biochrom, Berlin, Germany) and 100 U/ml penicillin/100 pg/ml streptomycinmixture (Invitrogen). ND7-23 cells (European Collection of Cell Cultures, PortonDown, UK) were cultured in DMEM (Invitrogen) with 10% fetal bovine serum,100 U/ml penicillin/100 pg/ml streptomycin mixture, and 2 mM L-glutamine(Invitrogen). Transient expression was performed with Lipofectamine 2000(Invitrogen) according to the manufacturer’s protocol.

The rats (body weight 100–120 g) were anesthetized and euthanized. The DRGswere dissected, digested with 1 mg/ml collagenase type 1A, 0.4 mg/ml trypsintype I, and 0.1 mg/ml DNase I (all Sigma) in DMEM (Invitrogen) at 37 C for30 min, and then triturated. The dissociated DRG neurons were transfected byelectroporation with Nucleofector II (Amaxa Biosystems, Cologne, Germany)using a nucleofection kit and then cultured in DMEM containing 10% fetal bovineserum. After 6 hours, the culture medium was replaced with DMEM/F12 (1:1)containing 1% N2 supplement (Life Technologies, Grand Island, NY), and theneurons were maintained for further experiments. To test the knockdownefficiency of IPP5 shRNA and Smad2 shRNA, the freshly isolated DRGneurons were harvested 48 hr after transfection. For biochemical assays to testthe regulation of IPP5 on the TGF-b/Smad signaling pathway, the dissociatedDRG neurons were incubated with 4 mM cytosine-1-b-D-arabinofuranoside toinhibit glial proliferation and transfected with control siRNA or IPP5 siRNA usingLipofectamineTM RNAiMAX reagent (Invitrogen) 18 hr later. The culture mediumwas replaced with DMEM/F12 (1:1) containing 1% N2 supplement after 6 hr, andthese neurons were harvested for immunoblotting after 48 h. The oligonucleotidesfor control siRNA and IPP5 siRNA were as follows: 59-uucuccgaacgugucacgutt-39

and 59-acgugacacguucggagaatt-39; 59-cccuugugauucucaaugatt-39 and 59-ucauugagaaucacaagggat-39.

Neurite growth detection

The dissociated DRG neurons were transfected by electroporation with theindicated plasmids together with the GFP plasmid (1:1) if necessary, plated onpoly-D-lysine (Sigma)-coated cover glasses, and fixed for imaging after 48–72 h.For each experiment, 30–50 neurons expressing GFP were selected randomly foreach group; however, nearly the same number of neurons were measured fromeach group for one independent experiment. The length of the longest neurite, thetotal neurite length, and the number of neurite ends per neuron were measured and

analyzed with Neurolucida software (MBF Bioscience, Williston, VT). The valueswere then averaged for each experiment, and the data were pooled from threeindependent experiments and normalized to the control. The cumulative frequencywas calculated from the proportion of the total events. For the representativeimages, we further converted the RGB images to grayscale, inverted, and adjustedthe contrast globally to get ones with black neurites and white background.

After electroporation, most of the large DRG neurons died; ,30% of thesurviving DRG neurons were transfected, but only ,80% of these transfectedneurons were IPP5 positive (supplementary material Fig. S2C,D). Therefore, forthe experiments using IPP5 shRNA or overexpression, ,20% of the transfectedDRG neurons in absent of endogenous IPP5 expression have been taken intoaccount which may have led to an underestimation of the effect on neurite growth.

Drug treatment

For the measurements of neurite growth, the DRG neurons were incubated with5 mM SB431542 (Santa Cruz Biotechnology); or 10 ng/ml human TGF-b1(Chemicon, Temecula, CA), TGF-b2 and TGF-b3 (Peprotech, Rocky Hill, NJ); or5 nM tautomycin (Calbiochem, Nottingham, UK) immediately after transfectionand maintained for 48 h. To detect the phosphorylation levels, co-IP efficiency andsubcellular localization, HEK293T cells and cultured DRG neurons were treatedfor 60 min with 10 ng/ml TGF-b1 48 h after transfection with control, IPP5 orIPP5 mutant plasmids. To detect the IPP5 phosphorylation, cultured DRG neuronswere treated for 10 min with 10 mM forskolin (Sigma) with or without 2 mMcyclosporin A (Sigma).

Immunoprecipitation and immunoblotting

Freshly isolated DRGs from adult rats or dissociated DRG neurons or transfectedHEK293T cells were lysed in ice-cold immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 10% glycerol).The lysate was immunoprecipitated with 0.5 mg of a mouse antibody against IPP5or HA tag (Sigma) or rabbit antibody against Flag tag (Sigma) and then incubatedwith protein G-Sepharose beads (Amersham Biosciences, Heidelberg, Germany).For immunoblotting, the lysates or beads were incubated in SDS-PAGE loadingbuffer. The samples were separated on an SDS-PAGE gel, transferred, and probedwith homemade antibodies against IPP5 (1:10000); PP1 (1:1000; Cell SignalingTechnology, Boston, MA); phosphorylated Akt ser473 (1:1000; Cell SignalingTechnology); phosphorylated CREB Ser133 (1:1000; Cell Signaling Technology);GAPDH (1:50,000; Abcam); Smad2/3 (1:5000; Santa Cruz Biotechnology); GFP(1:10,000; Roche, Burlington, NC); phosphorylated Thr34 of IPP5 (1:1000, CellSignaling Technology); or rabbit antibodies against phosphorylated GSK3b Ser9(1:1000; Cell Signaling Technology); GSK3b (1:1000; Cell SignalingTechnology); Akt (1:1000, Abcam); CREB (1:1000, Cell SignalingTechnology); phosphorylated Smad2/3 (1:10,000); TbRI (1:1000); HA tag(1:1000; Sigma); Flag tag (1:5000; Sigma). The immunoreactive bands werethen detected with horseradish peroxidase-conjugated secondary antibodies,visualized with enhanced chemiluminescence (Amersham Biosciences) andquantified with Image-Pro Plus software (Media Cybernetics Inc., Bethesda,MD). Each experiment was repeated at least three times.

Protein purification and in vitro phosphorylation

Briefly, Escherichia coli BL21 were transformed with constructs encoding GST-fused IPP5 or IPP5T34D, and protein production was induced with 0.1 mMisopropyl-b-D-thiogalactopyranoside (Amresco, Solon, OH). The fusion proteinswere loaded onto a column packed with glutathione-Sepharose beads (AmershamBiosciences). Instead of eluting the fusion protein with glutathione buffer, asolution of 2.5 U/ml thrombin (Amersham Biosciences) was loaded onto thecolumn and allowed to incubate at room temperature for 3 h. After elution fromthe column, the reaction was terminated by adding 1 mM PMSF, heating in aboiling water bath for 5 min and centrifuging at 40,000 g for 20 min. Thesupernatant was collected and quantitatively analyzed by the Bradford assay(Sigma). For the measurement with autoradiograph, purified IPP5 or IPP5T34D

(200 ng) was suspended in 30 ml reaction buffer (50 mM Tris-HCl, pH 7.5 and10 mM MgCl2) and incubated with 200 mM ATP, 50 U purified PKA (NewEngland BioLabs, Ipswich, MA) and 5 mCi [c-32P]ATP (PerkinElmer, Waltham,MA) at 30 C for 20 min. The phosphorylation status was analyzed by SDS-PAGEand autoradiography. For the measurement with site-specific monoclonal antibodyagainst phosphorylated Thr34 of IPP5, purified GST-IPP5 (800 ng) was suspendedin 25 ml reaction buffer and incubated with 25 mM ATP and 100 U purified PKAwith or without 50 mM H89 (Calbiochem) at 30 C for 4 hr. Phosphorylation statuswas analyzed by SDS-PAGE and immunoblotting.

Statistical analyses

The data are shown as means 6 s.e.m. Statistical significance was calculated usingunpaired or paired Student’s t-tests. The Kolmogorov–Smirnov test (KS-test) wasperformed to determine the significance between two groups for the cumulativefrequency of the length of the longest neurite, the total neurite length and thenumber of neurite ends. Differences were considered significant at P,0.05.

Journal of Cell Science 126 (2)552

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AcknowledgementsWe thank Dr Yeguang Chen for providing the plasmids of FLAG-tagged Smad2 and Smad3, and HA-tagged TbRI.

FundingThis work was supported by grants from the National NaturalScience Foundation of China [grant number 30930044 to L.B.]; theNational Basic Research Program of China [grant number2010CB912001 to L.B.]; and the Strategic Priority ResearchProgram (B) of the Chinese Academy of Sciences [grant numberXDB01020300 to X.Z.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.114280/-/DC1

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