Immunity

Article

SOCS3 Protein Developmentally Regulatesthe Chemokine Receptor CXCR4-FAK SignalingPathway during B LymphopoiesisYi Le,1 Bing-Mei Zhu,2 Brendan Harley,1 Shin-Young Park,1 Takashi Kobayashi,3 John P. Manis,1

Hongbo R. Luo,1 Akihiko Yoshimura,3 Lothar Hennighausen,2 and Leslie E. Silberstein1,*1Children’s Hospital Boston and Joint Program in Transfusion Medicine, Harvard Medical School, Boston, MA, 02115, USA2Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, MD 20892, USA3Division of Molecular and Cellular Immunology, Kyushu University, Fukuoka, Japan

*Correspondence: leslie.silberstein@childrens.harvard.edu

DOI 10.1016/j.immuni.2007.09.011

SUMMARY

The chemokine CXCL12 induces prolongedfocal adhesion kinase (FAK) phosphorylationand sustained proadhesive responses in pro-genitor bone-marrow (BM) B cells, but not inmature peripheral B cells. Here we demonstratethat suppressor of cytokine signaling 3 (SOCS3)regulated CXCL12-induced FAK phosphory-lation through the ubiquitin-proteasome path-way. CXCL12 triggered increased FAK ubiquiti-nation in mature B cells, but not in progenitor Bcells. Accordingly, SOCS3 expression was lowin progenitor B cells, increased in immature Bcells, and highest in mature B cells. SOCS3overexpression in pro-B cells impaired CXCL12-induced FAK phosphorylation and proadhesiveresponses.Conversely,SOCS3-deficient matureB cells from CreMMTVSocs3fl/fl mice exhibitedprolonged FAK phosphorylation and adhesionto VCAM-1. In contrast to wild-type mice,CreMMTVSocs3fl/fl mice had a 2-fold increase inimmature B cells, which were evenly distributedin endosteal and perisinusoidal BM compart-ments. We propose that the developmentalregulation of CXCR4-FAK signaling by SOCS3is an important mechanism to control the lodge-ment of B cell precursors in the BM microenvi-ronment.

INTRODUCTION

Hematopoiesis is a highly regulated process whereby

hematopoietic stem cells (HSC) give rise to specific cell

lineages, of which developing B cells are among the

best characterized (Hardy, 2003; Hardy and Hayakawa,

2001). The differentiation into different cell lineages from

HSC occurs as a progression from primitive, multilineage

potential progenitors through more restricted progenitors

Im

(LeBien, 2000). Committed precursor B cells execute

a programmed development, commencing with heavy-

chain immunoglobulin (Ig) rearrangement at the pro-B

cell stage. Cytoplasmic IgM expression defines the next

stage as pre-B, and once light chain is expressed, the

pre-B cells progress to immature B cells expressing IgM

and mature B cells expressing IgM and IgD on their sur-

face. In the bone marrow (BM), B cell progenitors reside

in close contact with stromal cells in distinct compart-

ments, i.e., niches, to receive specific signals for growth

and maturation (Osmond et al., 1992). The earliest progen-

itor B cell stages reside close to the endosteal surface,

whereas more mature B cells are localized more centrally

in close proximity to the central sinus before they exit into

the peripheral circulation (Jacobsen and Osmond, 1990;

Osmond et al., 1992). The localization and migration

between BM niches of hematopoietic progenitor cells are

thought to depend on soluble, cell-associated, and cell-

matrix interactions involving the cooperation of cytokines,

chemokines, and adhesion molecules (Scadden, 2006).

Among the chemokines, CXCL12 has been shown to

play an essential role in B lymphopoiesis as documented

through studies with CXCL12- and/or CXCR4-targeted

mice (Ma et al., 1998; Nagasawa et al., 1996). However,

the mechanisms underlying its role in B cell development

are not clear.

CXCL12 could exert a direct proliferative effect on B cell

progenitors (Nagasawa et al., 1994) and/or have a posi-

tioning effect, thereby directing B cell progenitors to the

appropriate supportive niches in the BM (Ma et al.,

1999). This positioning effect could involve (1) chemoat-

traction of B lineage cells and/or (2) adhesion to BM stro-

mal and/or endothelial cell molecules, e.g., VCAM-1 and

extracellular matrix molecules (for example, fibronectin)

(LeBien, 2000). In the BM, CXCL12 is expressed in soluble

form and immobilized to reticular, endothelial, and osteo-

blast cell types as well as to components of the extracellular

matrix (Dar et al., 2005; Lane et al., 2000; Peled et al., 1999).

Thus, it can be envisioned that regulation of CXCL12-

induced responses involving migration and adhesive inter-

actions may influence the localization of B cell populations

in distinct BM niches. CXCL12 can trigger marked biologic

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Developmental SOCS3 Regulation of B Lymphopoiesis

responses (i.e., chemotaxis and Ca signaling) of early, pro-

B, and pre-B cells, whereas the same responses by more

mature B cells from BM are diminished or absent (Palme-

sino et al., 2006). The diminished CXCL12-induced respon-

siveness by more mature B cells is disproportionate to the

amount of fluorescence intensity of CXCR4 surfaceexpres-

sion (Honczarenko et al., 1999), suggesting that other

mechanisms regulate CXCR4 function during B cell devel-

opment.

We previously reported that in progenitor B cells,

CXCL12 triggers prolonged focal adhesion kinase (FAK)

phosphorylation correlating with sustained proadhesive

responses to VCAM-1 (Glodek et al., 2003). In follow-up

studies, we determined that CXCL12-induced proadhe-

sive responses in progenitor B cells are dependent on

FAK expression (Glodek et al., 2007). After CXCL12 stim-

ulation of progenitor B cells, a fraction of cytoplasmic FAK

is recruited to raft membrane domains and phosphory-

lated at FAK-Y397. Moreover, CXCL12-induced proadhe-

sive responses are impaired when FAK recruitment to the

plasma membrane and phosphorylation are inhibited by

either membrane cholesterol depletion or overexpression

of Regulator of G Protein Signaling 1 (Le et al., 2005). Thus,

taken together, these studies suggested the importance

of FAK signaling in regulating adhesive interactions of pro-

genitor B cells in the BM.

FAK is a ubiquitously expressed nonreceptor protein

tyrosine kinase, whose function has been well studied in

fibroblasts, where it plays an important regulatory function

in cell-matrix-dependent adhesion, survival, and motility

(Mitra et al., 2005; Tilghman et al., 2005). After integrin-

mediated binding to extracellular matrix proteins such as

fibronectin, FAK is catalytically activated and undergoes

autophosphorylation at Y397, which serves as a binding

site for Src-homology 2 (SH2) domain containing Src fam-

ily kinases. The FAK-Src kinase complex leads to further

phosphorylation at additional FAK sites and to the recruit-

ment and activation of multiple downstream signaling pro-

teins. FAK, however, is also expressed in other cell types

including neuronal, endothelial, and hematopoietic cells

(Beggs et al., 2003; Kapur et al., 2001; Shen et al., 2005;

Takahira et al., 1997). The mechanisms by which FAK is

activated in these different cell types are not well defined,

but several studies suggest that they differ in some as-

pects from activation pathways in fibroblasts (del Pozo

et al., 2004; Le et al., 2005; Palazzo et al., 2004). Although

much is known regarding FAK activation pathways in

fibroblasts, information on how these pathways are nega-

tively regulated is limited.

Recent studies in 3T3 fibroblasts, however, have impli-

cated suppressor of cytokine signaling (SOCS) proteins in

the attenuation of FAK signaling and FAK-dependent cell

motility on fibronectin (Liu et al., 2003). Although SOCS

proteins are best known for providing a feedback loop to

inhibit growth factor responses and activation of the janus

kinase (JAK) and signal transducer and activator of tran-

scription (STAT) pathway (Alexander, 2002; Kubo et al.,

2003), it is evident that they can regulate other signaling

pathways as well (Johnston and O’Shea, 2003; Naka

812 Immunity 27, 811–823, November 2007 ª2007 Elsevier Inc.

et al., 2005). SOCS proteins contain a N-terminal region,

a central SH2 domain, and a conserved C-terminal

SOCS box. The SH2 domain mediates binding to phos-

phorylated tyrosine residues on signaling proteins, and

the C-terminal SOCS box domain has been found to inter-

act with Elongin B-C, Cul-5, and Rbx1-2 to form an E3

ubiquitin ligase complex. Thus, the SOCS proteins also

may target tyrosine kinases for polyubiquitination and

degradation in the proteasome (Kobayashi et al., 2006;

Mansell et al., 2006).

Based on these studies, we theorized that SOCS

proteins also play a role in the negative regulation of

CXCL12-induced FAK activation and adhesive responses

in B lineage cells in the BM. We determined that in unsti-

mulated, freshly isolated B cells from BM, SOCS3, but

not SOCS1, expression increases with B cell differentia-

tion. SOCS3 expression is relatively low in progenitor B

cells and increases at the immature B cell stage

(IgM+IgD�) and highest in splenic B cells. After CXCL12

signaling, SOCS3 interacts with FAK, leading to polyubi-

quitination and subsequent degradation. This interaction

and its consequences occur prominently in IgM+, imma-

ture, and mature B cells. Thus, our studies provide a

mechanism whereby low amounts of SOCS3 expression

allow for lodging of progenitor B cells in BM niches for

prolonged periods for growth and differentiation,

whereas higher amounts of SOCS3 expression promote

the exit of immature B cells and the circulation of mature

B cells.

RESULTS

CXCL12 Induces Prolonged FAK Phosphorylationand Adhesion to VCAM-1 in Murine BM B CellsCXCL12 induces prolonged (up to 20 min) FAK phosphor-

ylation in human BM B cells but only transient (lasting up to

1–3 min) FAK phosphorylation in mature peripheral blood

B cells. Moreover, the duration of FAK phosphorylation

correlated with the duration of CXCL12-induced adhesion

to VCAM-1 (Glodek et al., 2003). To extend our findings to

the murine system, we tested the pattern of CXCL12-

induced FAK phosphorylation in B cells from BM and

spleen of 8-week-old C57/BL6 mice. CXCR4 expression

is comparable on murine BM and splenic B cells (Figure S1

in the Supplemental Data available online). As shown in

Figure 1A, CXCL12 induces strong FAK phosphorylation

in murine BM B cells lasting up to 15 min. In contrast,

mature splenic B cells exhibit only weak and transient

(1 min) FAK phosphorylation after CXCL12 stimulation.

BM B cells were then sorted by flow cytometry into early

B lineage, immature, and mature B cells. We found that

CXCL12 triggers increased FAK phosphorylation in early

B lineage cells compared to late B lineage cells (Figure 1B).

Also, CXCL12-induced adhesion is strongest in early B

lineage cells and diminishes with maturation (Figure 1C).

Thus, these findings show that in both mice and humans,

CXCL12 triggers enhanced FAK phosporylation and

adhesion to VCAM-1 in progenitor BM B cells compared

to mature peripheral B cells.

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Developmental SOCS3 Regulation of B Lymphopoiesis

Figure 1. CXCL12 Induces Prolonged FAK Phosphorylation and Adhesion to VCAM-1 in Murine BM B Cells, but Not in Spleen B Cells

(A) B cells from mouse BM and SP were stimulated with 100 nM CXCL12 for indicated times.

(B) Mouse BM cells were sorted into early B lineage (early B; B220+IgM�), immature B lineage (IMB; B220+IgM+IgD�), and mature B (MB;

B220+IgM+IgD+) cell populations. Isolated B cells were stimulated with CXCL12 for 5 min. Cell lysates were analyzed by immunoblotting (IB) with

phospho-FAK Abs and FAK Abs. Numbers under each lane are based on densitometry values and indicate the increase of phosphorylated FAK

expressed as multiple of control (assigned as 1.0).

(C) Total mouse BM and SP lymphocytes were subjected to an adhesion assay to VCAM-1. Adherent cells were then retrieved by EDTA treatment and

stained with Abs recognizing surface antigens that define the following B cell maturation stages: early B, IMB, and MB cells. Data are representative of

three independent experiments, each performed in triplicate. * and **, statistical significance as compared with early B lineage cells and are assessed

as p < 0.05 and p < 0.01, respectively.

CXCL12 Induces FAK Ubiquitination in MaturePeripheral B Cells, but Not in ProgenitorBM B CellsLigand-induced ubiquitination is one of several mecha-

nisms, which can modulate the amount and duration of

signaling activity of protein tyrosine kinases such as Syk

and Zap-70 in lymphocytes (Paolini et al., 2001; Sohn

et al., 2003). Therefore we questioned whether ubiquiti-

nation might similarly regulate CXCL12-mediated FAK

phosphorylation. We examined FAK ubiquitination and

phosphorylation in BM and mature B cells after 10 min

of CXCL12 stimulation. Multiple ubiquitinated FAK bands

ranging in size from 130 kDa to 210 kDa were generated

in mature peripheral B cells, whereas these bands were

substantially less detectable in CXCL12-stimulated total

BM B cells, as shown by the fact that they contain approx-

imately 50% pro-B and pre-B cells (Figure 2A). At the

same time, we determined that 10 min of CXCL12 stimu-

lation induced increased amounts of FAK phosphorylation

in BM B cells compared to peripheral B cells (Figure 2A),

consistent with our previous results (Glodek et al., 2003).

Because substantially more cells are required for time-

course studies, we utilized a pro-B cell line, REH, and

a mature B cell line, HS-Sultan. We found that in HS-Sul-

tan cells, ubiquitinated FAK bands were observed after

1 min of CXCL12 stimulation, reached a peak at 10 min,

and began to decrease at 30 min of CXCL12 stimulation

(Figure 2B). Importantly, increased FAK ubiquitination

correlated with transient FAK phosphorylation in CXCL12-

Im

stimulated HS-Sultan cells. In contrast, barely detectable

ubiquitinated FAK bands in REH pro-B cells upon CXCL12

stimulation correlated with prolonged FAK phosphoryla-

tion and adhesion to VCAM-1 (Figure 2B). Because plate-

let-derived growth factor-induced FAK ubiquitination in

fibroblast cells is dependent on its phosphorylation at

tyrosine 397 (FAK-PY397) (Liu et al., 2003), we examined

the effect of Pertussis toxin (PTX), an inhibitor of Gia pro-

tein and CXCL12-mediated FAK phosphorylation (Glodek

et al., 2003; Le et al., 2005), on CXCL12-induced FAK

ubiquitination in lymphocytes. PTX treatment of HS-Sultan

cells, which were stimulated for 10 min by CXCL12,

impairs CXCL12-induced ubiquitination, implying that

CXCL12-induced FAK ubiquitination occurs after Gia sig-

naling (Figure 2C). Taken together, these results demon-

strate an inverse relationship between CXCL12-induced

FAK phoshorylation and FAK ubiquitination during B cell

differentiation.

The Proteasome Inhibitor MG132 ProlongsCXCL12-Induced FAK Phosphorylationin Mature Hs-Sultan B CellsA prominent role of ubiquitination is to target phosphory-

lated proteins for degradation in the proteasome. In this

regard, we found that treatment of HS-Sultan cells with

the proteasome inhibitor, MG132 (Calbiochem), increased

the duration of phospho-FAK from 3 min to 30 min

(Figure 3A), suggesting that MG132 treatment inhibited

degradation of phospho-FAK. For confocal microscopic

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Developmental SOCS3 Regulation of B Lymphopoiesis

analysis, cells were stained with a phospho-FAK(PY397)

antibody and cholera toxin (CTX) to detect the lipid raft

component GM1. In unstimulated control cells, phos-

pho-FAK(PY397) was not detectable (Figure 3B). After

CXCL12 stimulation for 3 min, we noticed that phospho-

FAK(PY397) staining was distributed primarily near the

cell membrane in both MG132-treated and DMSO-treated

cells, although with increased intensity in MG132-treated

cells (Figure 3B). However, after CXCL12 stimulation for

10 min, phospho-FAK was no longer seen in DMSO-

Figure 2. CXCL12 Induces Prolonged FAK Phosphorylation

Correlating with Weak FAK Ubiquitination in Progenitor

B Cells; in Contrast, CXCL12 Triggers Transient FAK Phos-

phorylation Correlating with Increased FAK Ubiquitination in

Mature B Cells

(A) Human and mouse BM and peripheral blood (PB) or SP B cells were

stimulated with 100 nM CXCL12 for 10 min.

(B) Pro-B cell line REH and mature B cell line HS-Sultan were stimu-

lated with 100 nM of CXCL12 for indicated times, followed by immuno-

precipitation (IP) and IB. Representative results from one of three inde-

pendent experiments are shown.

(C) PTX reduces CXCL12-induced FAK ubiquitination. HS-Sultan cells

were incubated with 100 ng/ml of PTX for 2 hr, followed by CXCL12

stimulation, IP, and IB. Representative data from one of three experi-

ments are shown.

814 Immunity 27, 811–823, November 2007 ª2007 Elsevier Inc.

treated cells, consistent with immunoblotting (IB) analysis

in Figure 3A. Moreover, 10 min of CXCL12 stimulation in-

duced clustering of lipid rafts (Le et al., 2005), and we were

able to observe colocalization of phospho-FAK and the

raft marker GM1 as indicated by the yellow margin in

MG132-treated cells but not in DMSO-treated cells. These

results support the idea that after CXCL12 stimulation,

phosphorylated FAK is ubiquitinated and targeted for deg-

radation in the proteasome.

SOCS3 Expression Increases during B CellDevelopment and Coassociates with FAKupon CXCL12 StimulationTo examine SOCS1 and SOCS3 expression in B cells rep-

resenting early and late stages of differentiation, we iso-

lated early (pro- and pre-) and late (immature and mature)

B cells from human BM and CD19+ B cells from peripheral

blood. By RT-PCR, SOCS3 expression was low in early B

lineage cells and was increased in late B cells (Figure 4A).

SOCS3 amounts were highest in peripheral B cells

(Figure 4A). In contrast, there was no difference in the

expression of SOCS1 and FAK in any of the three B cell

populations tested. The RT-PCR results were also con-

firmed by IB analysis. Similarly, SOCS3 protein expression

is increased in mature B cells and the mature B cell line

HS-Sultan, whereas SOCS1 protein expression is low in

all three B cell populations (Figure 4B). Next, we deter-

mined that CXCL12 stimulation induces the coassociation

of FAK and SOCS3 in human peripheral blood B cells. A

weak association of FAK and SOCS3 also is observed in

unstimulated cells (Figure 4C). Collectively, these findings

suggested that SOCS3 may play an important role in the

developmental regulation of CXCR4-FAK signaling during

B cell differentiation.

SOCS3 Attenuates CXCR4-FAK Signalingand Cellular ResponsesBased on our findings thus far, we theorized that SOCS3

functions as a negative regulator of FAK signaling in mature

B cells. To test this hypothesis, we used the pro-B cell line

REH to establish stable expression of GFP-SOCS3, and

we used control vector GFP as a control. We also stably

expressed a mutant GFP-SOCS3 gene lacking the SOCS

box region (referred to as GFP-mSOCS3). The transfected

cell lines containing 90% GFP-positive cells are shown

in Figure 5A. Overexpression of GFP-SOCS3 caused a

reduction in CXCL12-induced FAK phosphorylation and

an increase in CXCL12-induced FAK polyubiquitination

(Figure 5B). In contrast, overexpression of GFP-mSOCS3

did not exhibit these effects, indicating that the SOCS

box of SOCS3 is important for attenuating the CXCR4-

FAK signaling pathway. Moreover, overexpression of

SOCS3 impaired CXCL12-induced REH cell chemotaxis

and adhesion to VCAM-1 (Figures 5C and 5D), whereas

mutant, SOCS box-deficient SOCS3 did not exhibit such

effects.

Finally, we examined whether SOCS3 overexpression

inhibits CXCL12-induced cell polarization. Unstimulated

GFP-REH cells were predominantly round (Figure 5E;

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Developmental SOCS3 Regulation of B Lymphopoiesis

Figure 3. MG132 Prolongs CXCL12-Induced FAK Phosphorylation in Mature Hs-Sultan B Cells

(A) Hs-Sultan cells were treated with 5 mM MG132 or DMSO as control and then stimulated with CXCL12 for the indicated times. Samples were

analyzed by IB with phospho-FAK (PY397) and total FAK Abs. Representative results from one of three independent experiments are shown.

(B) MG132-treated or not treated cells were stimulated with CXCL12 for 0, 3, 10 min at 37�C. Cells were then labeled with Alexa 488-conjugated CTX

(green color). Subsequently, cells were fixed, permeabilized, and stained with a phospho-FAK (PY397) Ab, followed by an Alexa 568-conjugated

secondary Ab (red color). Cells were then mounted on glass slides and analyzed by confocal microscopy. Cells in each image field are representative

of at least 60 cells.

Movie S1). However, when uniformly stimulated with

CXCL12, GFP cells displayed membrane ruffles and

polarized, forming distinct pseudopods and uropods

(Figure 5E; Movie S2). In contrast, GFP-SOCS3 cells did

not display membrane ruffles, nor did they polarize upon

CXCL12 stimulation (Figure 5E; Movie S3). Furthermore,

GFP-mSOCS3 cells behaved similar to GFP cells in that

I

they also polarized upon CXCL12 stimulation (Figure 5E;

Movie S4). When we quantified the fraction of ruffling cells,

there is no significant difference between GFP-mSOCS3

and GFP cells (p > 0.05) (Figure 5F), further implicating

the SOCS box in SOCS3 protein regulatory function.

Thus, these experiments provide evidence for the nega-

tive regulation by SOCS3 of CXCR4-FAK signaling.

Figure 4. Increased Expression of SOCS3, but Not SOCS1 during B Cell Development

(A) SOCS3, SOCS1, and FAK mRNA expression was determined by RT-PCR of mRNA extracted from human BM early and late B cells and peripheral

B cells. GAPDH-normalized mRNA is indicated as the fold difference among B cell populations relative to mRNA expression from early BM B cells. The

developmental stage of each B cell population is indicated. Data represent the mean ± SD of three experiments, each performed in triplicate. * and **,

statistical significance and are assessed as p < 0.05 and p < 0.01, respectively.

(B) 2 3 106 of BM early B, late B, and PB cells as well as the pro-B REH and the mature B cell line, HS-Sultan were lysed and 1/3 of total lysates were

used for IB.

(C) CXCL12-induced association of FAK and SOCS3 in human peripheral blood B cells. Stimulated and unstimulated cells were IP with a FAK or

SOCS3 Ab. Samples were analyzed by IB. Representative results from one of three independent experiments are shown.

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Developmental SOCS3 Regulation of B Lymphopoiesis

Figure 5. Overexpression of SOCS3 Inhibits CXCL12-Induced FAK Phosphorylation and Cellular Responses

(A) Similar amounts of gene expression in transfected REH cells. GFP-SOCS3, GFP-mSOCS3, or GFP expression vectors were transfected into REH

cells and sorted by GFP expression.

(B) After CXCL12 stimulation (100 nM), transfected cells were subjected to IP and IB analysis. Endogenous SOCS3 expression was assessed by IB

analysis of total cell lysate (TCL).

(C) Transwell chemotaxis of transfected cells toward CXCL12 (10 nM). Data are shown as the percent of specific migration. Mean ± SD of three

independent experiments done in duplicate is shown.

(D) Transfected REH cells were subjected to an adhesion assay to VCAM-1. The data shown are the mean ± SD from five separate experiments.

*p < 0.01 versus GFP cells.

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Developmental SOCS3 Regulation of B Lymphopoiesis

Figure 6. Enhanced B Cell Responses to CXCL12 in CreMMTVSocs3fl/fl Mice

(A) Quantitative RT-PCR was performed on mRNA extracted from B cell subsets. GAPDH-normalized mRNA expression is indicated as the difference

relative to mRNA expression of BM early B cells of Socs3fl/fl mice. Data are mean ± SD collected from three independent experiments. * and **, sta-

tistical significance between two groups of mice and are assessed as p < 0.05 and p < 0.01, respectively. In Socs3fl/fl mice, the difference between

early B lineage and immature B (IMB) cells is statistically significant (p < 0.05) as well as between early B and mature B (MB) cells (p < 0.01).

(B) SOCS3 expression in B cell subsets.

(C) CXCL12 induces prolonged FAK phosphorylation in SOCS3-deficient B cells, but not in control B cells. Spleen B cells were stimulated with

CXCL12 for indicated times and IB as described in Figure 1A.

(D) CXCL12-induced adhesion to VCAM-1 is increased in murine SOCS3-deficient B cells compared to B cells from control mice. The adhesion assay

was performed as described in Figure 1C. Data are mean ± SD collected from three independent experiments. * and **, statistical significance

between two groups of mice and are assessed as p < 0.05 and p < 0.01, respectively.

Increased Cellular Responses to CXCL12in SOCS3-Deficient B CellsTo further define SOCS3 function, we examined B cells

from CreMMTVSocs3fl/fl mice, in which Cre is expressed

under the control of the mouse mammary tumor virus

(MMTV) long terminal repeat (LTR). The MMTV-Cre trans-

gene in these mice is active in the hematopoietic compart-

ment including T and B cells (Wagner et al., 2001). First we

established that Socs3 gene is efficiently deleted in BM

and splenic B cells of CreMMTVSocs3fl/fl mice by PCR gen-

otyping (Figure S2), quantitative RT-PCR on total RNA

(Figure 6A), and IB analysis (Figure 6B). Additionally, we

demonstrated that similar to human BM B cells, SOCS3

expression is relatively low in early progenitor B220+IgM�

B cells, increases in immature, IgM+IgD� B cells, and is

Im

highest in peripheral mature IgM+IgD+ B cells (Figures

6A and 6B). Subsequently, we compared the duration

and intensity of CXCL12-induced FAK phosphorylation

in B cells from CreMMTVSocs3fl/fl mice and Socs3fl/fl wild-

type mice lacking Cre. Compared to wild-type B cells,

SOCS3-deficient B cells exhibit increased and prolonged

FAK phosphorylation (Figure 6C), which correlated with

decreased FAK ubiquitination (Figure S3). These findings

indicate that SOCS3 negatively regulates CXCL12-

induced FAK phosphorylation in B cells. In contrast,

SOCS3 deficiency did not affect CXCL12-induced phos-

phorylation of MAP kinase.

Because FAK phosphorylation is necessary for CXCL12-

induced adhesion to VCAM-1 (Le et al., 2005), we specu-

lated that SOCS3 might also affect this CXCL12-mediated

(E) Transfected REH cells were plated on Labtek chamber slides and uniformly stimulated with 200 nM of CXCL12 or without stimulation. After 1 min of

stimulation, time-lapse images were recorded for 10 min.

(F) Percentage of cells that ruffled or extended pseudopods was calculated from images captured 4 to 8 min after CXCL12 stimulation. Data are mean ±

SD collected from three separate experiments. *p < 0.01 versus GFP-vector cells. Four videos of this experiment are also included in Movies S1–S4.

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Developmental SOCS3 Regulation of B Lymphopoiesis

Figure 7. Accumulation of Immature B Cells in CreMMTVSocs3fl/fl Mice Compared to Socs3fl/fl Mice

(A) BM of CreMMTVSocs3fl/fl and Socs3fl/fl mice were stained with indicated Abs, followed by FACS analysis. Gating region R1 includes

B220+IgM+IgD�; R2 includes B220+IgM+IgDlow; R3 includes B220+IgM+IgDhigh.

(B) 2-fold increase in IgM+IgD� B cell number in BM of CreMMTVSocs3fl/fl mice. Cell numbers were calculated from total number of BM cells multiplied

by the percentage of corresponding B lineage cells. Statistically significant results were calculated by Student’s t test: *p < 0.01; n = 8 mice in each

group.

(C) Distribution of IgM+ cells across the entire bone cross-section (103 magnification), at the endosteum, and the central region of the marrow

(403 magnification). Frozen sections derived from the femurs of CreMMTVSocs3fl/fl and Socs3fl/fl mice were stained with anti-IgM (red). Data are rep-

resentative of two mice in each group.

(D) The graph represents the mean fluorescence intensity of IgM+ staining, expressed as mean ± SD from seven images of each region (403 mag-

nification). *, statistical significance between endosteal region and central sinus of Socs3fl/fl mice and is assessed as p < 0.01.

cellular response. In Socs3fl/fl wild-type B cells, CXCL12-

induced adhesion to VCAM-1 diminishes during B cell

maturation and is absent in mature peripheral B cells

(Figure 6D). In contrast in mature B cells of SOCS3-defi-

cient mice, CXCL12 induced enhanced adhesion to

VCAM-1 correlating with prolonged FAK phosphorylation

(Figure 6D). CXCR4 expression was unaffected by SOCS3

deficiency (Figure S1). Thus, in the absence of SOCS3, ma-

ture B cells exhibit CXCL12-induced responses similar to

progenitor B220+IgM� B cells.

818 Immunity 27, 811–823, November 2007 ª2007 Elsevier Inc

Accumulation of Immature B Cells in BMof CreMMTVSocs3fl/fl MiceTo evaluate whether SOCS3 deficiency might differentially

affect stage-specific B cells, BM B cells from CreMMTV

Socs3fl/fl mice and Socs3fl/fl mice were stained with Abs

to B220, AA4.1, IgM, and IgD. Remarkably, the percent-

age and absolute cell number of BM IgM+IgD� immature

B cells in CreMMTVSocs3fl/fl mice was 2-fold higher than

in control Socs3fl/fl mice (Figure 7). Moreover, the number

of B220+IgM+ cells, which expressed high amounts of

.

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Developmental SOCS3 Regulation of B Lymphopoiesis

AA4.1, a cell-surface marker of immature and transitional

B cells (Allman et al., 2001; Rolink et al., 1998), was also

increased in CreMMTVSocs3fl/fl mice. The SOCS3-deficient

IgM+IgDlow cell number also was slightly increased, but

the difference from control mice was not statistically sig-

nificant. The number of IgM+IgDhigh B cells, which are

mostly recirculating naive B cells, was similar in the two

groups of mice. There was also no difference in the num-

ber of progenitor B220+IgM� B cells (data not shown),

total BM B220+ cells (Figure 7B), and total BM cells

(data not shown). With respect to the mature B cell popu-

lation in spleen, the total number of B220+ and IgM+IgD+

cells tended to be lower in CreMMTVSocs3fl/fl mice com-

pared to Socs3fl/fl mice. However, the difference was

not statistically significant between the two groups

(Figure S4).

We theorized that the 2-fold increase of immature B

cells in BM of CreMMTV Socs3fl/fl mice could be due to en-

hanced adhesive interactions of IgM+ B cells with the BM

microenvironment, thus slowing or impeding their exit into

the periphery. We asked whether SOCS3 might also affect

the localization in BM of IgM+ B cells, which in wild-type

mice are located predominantly perisinusoidally (Cariappa

et al., 2005). This question prompted us to perform immu-

nofluorescence staining of BM sections followed by con-

focal microscopy. To approximate the number of IgM+

cells from the fluorescently labeled histological sections,

a measurement of mean fluorescent intensity (MFI) within

a field of view encompassing either endosteal region or

central sinus region (for definition see Experimental Proce-

dures) was made from sections stained and imaged under

constant conditions. In Socs3fl/fl mice, a statistically signif-

icant higher amount of IgM staining was observed near the

central sinus compared to the endosteal region (p < 0.01)

(Figures 7C and 7D). In contrast, in CreMMTVSocs3fl/fl mice

there was no difference of IgM staining between the two

regions (Figures 7C and 7D). These results indicate that

SOCS3 action influences the distribution of IgM+ cells in

the BM compartment.

DISCUSSION

In the BM, developing B cell populations reside in distinct,

albeit not well defined, microenvironments, i.e., niches

(Jacobsen and Osmond, 1990). As B cells differentiate,

they are thought to move from one niche to the next before

they egress at the immature-mature B cell stage into

the central sinus and peripheral circulation (Cariappa

et al., 2005; Nagasawa, 2006). However, the mechanisms

underlying B cell lodgement in niches, their migration

between niches, and ultimately their exit are not under-

stood. Based on previous studies, we have proposed

that the CXCR4-FAK-VLA4 pathway is important for sus-

tained adhesive interactions between progenitor B cells

and the BM microenvironment (Glodek et al., 2003, 2007;

Le et al., 2005). In the present study, we sought to under-

stand how this pathway might be regulated at different

stages of B cell development. Collectively, the data pre-

Im

sented here provide strong evidence for the negative reg-

ulation by SOCS3 of CXCR4-FAK signaling in B cells.

There are several potential mechanisms by which

SOCS3 may attenuate FAK signaling and activation. The

SOCS family members contain three domains, which

can potentially regulate FAK function. First, the amino

terminus of SOCS3 (and SOCS1) has a kinase inhibitory

region (KIR) (Nicholson et al., 1999), which can directly

inhibit JAK tyrosine kinase activity and thus may similarly

affect FAK kinase activity. Second, the SH2 domain of

SOCS proteins mediates binding to FAK-PY397 (Liu

et al., 2003), which could interfere with subsequent bind-

ing of Src kinase and thus inhibit further FAK phosphory-

lation and full activation of FAK. Finally, the C-terminal

SOCS box domain may interact with an E3 ligase complex

(Hilton et al., 1998), thus targeting FAK for ubiquitination

and degradation in the proteasome. We explored the latter

possibility by overexpressing a mutant form of SOCS3, in

which the SOCS box region was completely deleted. We

found that the mutant form of SOCS3 does not mediate

FAK ubiquitination and does not inhibit CXCL12-induced

chemotaxis or adhesion to VCAM-1. Thus, the attenuation

of CXCL12-induced FAK signaling in B cells is mediated

through the SOCS box region by targeting FAK for degra-

dation. However, SOCS3 also may regulate CXCL12-

induced cellular responses by attenuating other signaling

molecules downstream of CXCR4. For example, whereas

SOCS3 is expressed at low amounts in adult tissues, its

expression can be upregulated by growth hormone and

a number of cytokines, thus implicating SOCS3 in the neg-

ative feedback of cytokine signaling (Alexander, 2002;

Johnston and O’Shea, 2003). In this regard, growth hor-

mone-induced SOCS3 expression leads to blockade of

JAK and Gi protein association with CXCR4 and impaired

chemotaxis (Garzon et al., 2004; Pello et al., 2006; Soriano

et al., 2003). In addition, heat shock protein has been

reported to regulate CXCR4 signaling in T cells via a similar

mechanism (Zanin-Zhorov et al., 2005).

Remarkably, both in mouse and human, SOCS3 ex-

pression is upregulated during B lymphopoiesis. One pos-

sibility is that as B cells differentiate and localize in their

stage-specific environments, they are subject to niche-

specific cytokine and chemokine signaling that regulate

SOCS3 expression. In one attractive model, pre-pro

B cells associate with CXCL12-expressing stromal cells,

whereas pro-B cells adjoin IL-7-expressing stromal cells

(Tokoyoda et al., 2004). As B cells mature further, they

localize in other compartments of the BM and associate

with other cell types, e.g., immature B cells in contact

with Thy-1dull, DX5pos cells (Sandel et al., 2001). In contrast

to early precursor, IgM� B cells, which are thought to re-

side in close proximity to the endosteum, the more mature

IgM+ B cells in the BM are localized more centrally in peri-

sinusoidal compartments. Because SOCS3 upregulation

occurs at the IgM+ immature B cell stage and is highest

in peripheral IgM+IgD+ cells, it is attractive to speculate

that the mature B cell populations are in contact with dis-

tinct cell types, which could be the source of cytokines

such as IL-6, known to induce SOCS3 expression.

munity 27, 811–823, November 2007 ª2007 Elsevier Inc. 819

Immunity

Developmental SOCS3 Regulation of B Lymphopoiesis

Alternatively, other signaling pathways may control

SOCS3 expression, e.g., downstream of the immunoglob-

ulin receptor, either via ligand interaction with the surface

receptor or cell intrinsically through basal immunoglobulin

signaling (Tze et al., 2005).

The developmental regulation of SOCS3 expression is

of considerable interest. It is conceivable that the low

amount of SOCS3 expression in progenitor B cells favors

the action of growth factors, e.g., stem cell factor, IL-7,

and CXCL12, which are critical for early stages of B cell

development in BM. The upregulation of SOCS3 expres-

sion may be of importance to subsequent B cell differen-

tiation for several reasons. One possibility is that

increased SOCS3 action promotes B cell differentiation

by attenuating the JAK-STAT pathway downstream of the

aforementioned cytokines and chemokines. A second

mechanism of SOCS3 action in B lymphopoiesis is pro-

vided by studies presented here. By negatively regulating

the CXCR4-FAK pathway, SOCS3 fine-tunes the lodge-

ment of IgM+ B cells in the BM and facilitates their exit

into the peripheral circulation. Consistent with this idea

is the 2-fold increase in the number of IgM+IgD� immature

B cells inSOCS3-deficientmice, which wepropose isdue to

prolonged lodging of SOCS3-deficient immature B cells in

the BM. Moreover, SOCS3 may also affect the localization

of immature B cells because IgM+ B cells in wild-type

mice are localized predominantly near the central sinus,

whereas IgM+ B cells from SOCS3-deficient miceare evenly

distributed, both near the endosteum as well as near the

central sinus.Thisobservation argues thatnewly formed im-

mature B cells may arise from pre-B cells in endosteal

niches and subsequently migrate to the central sinus region.

In view of the current data and previous studies (Glodek

et al., 2003; Le et al., 2005), we propose a provisional

model to explain the roles of the CXCL12-CXCR4 axis

and SOCS3 in B cell development. CXCL12, which is pro-

duced by stromal cells, binds and activates CXCR4 on

progenitor B cells. Activated CXCR4 receptors form clus-

ters in membrane domains (van Buul et al., 2003) and sub-

sequently trigger signaling pathways, involving JAK and

STAT proteins, Gi proteins, Src family proteins, PI3-

kinase, FAK, and Rap1 (Glodek et al., 2007; Le et al.,

2005; Pello et al., 2006). Progenitor B cell surface integ-

rins, such as VLA-4, are activated and bind to both cell-

associated adhesion molecules, e.g., VCAM-1, as well as

extracellular matrix proteins, e.g., fibronectin. Sustained

adhesive interactions retain progenitor B cells in the BM

microenvironment (Ma et al., 1999) and promote progeni-

tor B cell growth, survival, and differentiation. During sub-

sequent B cell differentiation, however, CXCL12-induced

signaling and cellular responses are downregulated,

which is attributed in part to SOCS3 action. As a result,

proadhesive interactions between IgM+ B cells and their

microenvironment are diminished, thereby enabling their

exit into the periphery. The observation that the expres-

sion of SOCS3 as opposed to SOCS1 is upregulated at

the immature-mature B cell stage suggests a niche and/

or differentiation stage-specific function of SOCS3 during

B lymphopoiesis.

820 Immunity 27, 811–823, November 2007 ª2007 Elsevier In

EXPERIMENTAL PROCEDURES

Antibodies

mAbs against phosphotyrosine (4G10) were provided by T. Roberts

(Dana-Farber Cancer Institute, Boston, MA). Antisera against FAK

(A17), SOCS1 (H93), SOCS3 (M20), ubiquitin (P4D1), and c-Myc

(A14) were from Santa Cruz Biotechnology (Santa Cruz, CA); MAPK

and phospho-MAPK Abs were from Cell Signaling Technology (Dan-

vers, MA); SOCS3 Ab was from IBL (Gunma, Japan); HRP-conjugated

goat anti-mouse and goat anti-rabbit secondary Abs were from Bio-

Rad; conjugated anti-murine B220, or IgM, or IgD, or AA4.1 Abs

were from BD PharMingen. Immunofluorescence experiments were

performed with FAK (PY397) Abs, Alexa 568-conjugated goat-anti-

rabbit IgG, Alexa 488-conjugated goat-anti-mouse IgM, and Alexa

488-conjugated cholera toxin subunit B (Invitrogen, Carlsbad, CA).

Mice

Generation of mice with a conditional Socs3 allele has been described

previously (Yasukawa et al., 2003). These mice were mated with

a transgenic line bearing Cre recombinase driven by MMTV. Littermate

mice were generated homozygous for the conditional Socs3 alleles.

Animals were handled and housed in accordance with the guidelines

of the National Institutes of Health and the Children’s Hospital Boston

Animal Care and Use Committee.

Human B Cells

Human pro-B REH and lymphoblast HS-Sultan cells (American Type

Culture Collection) were maintained in RPMI 1640 supplemented

with 10% FBS, 1% penicillin and streptomycin, and 2 mM glutamine

(Invitrogen). Human B cell subsets were isolated from BM and periph-

eral blood from adult volunteers in accordance with guidelines

approved by the institutional review committees of the Dana-Farber

Cancer Institute and Children’s Hospital Boston. The early B lineage

cell population included both pro-B and pre-B cell subsets (CD19+,

kappa and lambda chain negative), whereas the late B lineage cell

population included immature and mature B cells (CD19+, kappa and

lambda chain positive) (Honczarenko et al., 2002). Peripheral B cells

were stained and sorted with a CD19 antibody and were regarded

as mature B cells.

Gene Transfection

Construct of pcDNA3-Myc-SOCS3 gene was described before (Yasu-

kawa et al., 2003). Mutant Myc-mSOCS3 was generated by deleting

C-terminal SOCS box gene. SOCS3 and mSOCS3 genes were then

subcloned into eGFP vector by restriction enzyme EcoR1 and Xho1.

For stable transfection, GFP-SOCS3, GFP-mSOCS3, and GFP vectors

were linearized by digestion with SalI (New England Biolabs) and trans-

fected into REH cells by electroporation. Neomycin-resistant clones

were analyzed for GFP expression by FACS. For analyses, a minimum

of 10 positive clones from each stably transfected cell line were pooled

and sorted by GFP expression on MoFlo cytometer (DakoCytomation,

Denmark) to establish comparable expression levels (Chang et al.,

2005).

Immunoprecipitation and Immunoblotting

Immunoprecipitation and immunoblotting were performed by previ-

ously described methods (Glodek et al., 2003; Le et al., 2005).

Immunofluorescence Microscopy of Progenitor B Cells

Immunofluorescence microscopy of progenitor B cells has been

reported previously (Le et al., 2005).

Immunohistology of BM Sections

Femurs from adult mice were frozen in OCT (Sakura Finetek, Torrance,

CA), longitudinally sectioned at a 4 mm thickness, and briefly fixed in

acetone. Sections were stained with an anti-IgM-Cy3 antibody (Jack-

son ImmunoResearch, West Grove, PA). Isotype control slides were

stained with Alexa Fluor 546 (Invitrogen) without the primary antibody.

c.

Immunity

Developmental SOCS3 Regulation of B Lymphopoiesis

All slides were then labeled with 100 nM DAPI (Invitrogen) and

mounted with Gel/Mount (Biomeda Corp., Foster City, CA). Confocal

microscopy was performed with a Zeiss Axoplan microscope and an

LSM 510 META confocal array. Postimaging processing was per-

formed with the LSM Image Analysis suite of programs (Zeiss, Ger-

many). For analysis of the relative distribution of IgM+ cells in the end-

osteal versus central sinus niche, the endosteal region was defined as

the area of cells within 15–20 cell diameters of the endosteal surface.

The central sinus region was defined as the center-most region of the

BM and was measured to be 50–75 cell diameters from the endosteal

surface (Yang et al., 2007).

Chemotaxis and Cell-Adhesion Assays

Chemotaxis assays were performed by previously described methods

(Honczarenko et al., 1999). Migratory cells were collected and counted

by timed acquisition (60 s/sample) on a flow cytometer. Cell adhesion

assays were performed as previously described (Glodek et al., 2003).

In brief, cells were stimulated with 1.0 mM CXCL12 in suspension at

37�C for 15 min and then placed into VCAM-1-coated wells for

30 min at 37�C. For REH cell adhesion, the number of bound cells

was determined with a CyQuant Cell Proliferation Assay kit (Molecular

Probes). For adhesion of murine BM and spleen cells, the adherent

cells were detached from the bottom of the wells by treatment with

0.01% EDTA in PBS and then stained with Abs to define early B lineage

(B220+IgM�), immature B (B220+IgM+IgD�), and mature B (B220+IgM+

IgD+) cells. Samples were analyzed on a Cytometer by timed acquisi-

tion, and the percentage of adhered cells within each population was

calculated in relation to the input control.

Cell Polarization Assay

REH cells were allowed to settle down for 5 min on Labteck cham-

bered coverglass and then stimulated with CXCL12 at a final concen-

tration of 200 nM. After 1 min stimulation, Nomarski/DIC was captured

with a 403 oil immersion objective on an Olympus IX-71 microscope

(Melville, NY). Images were acquired every 10 s for 10 min with

a CCD camera controlled by the IPLab imaging software (IPLab, Rock-

ville, MD). The percentage of polarized REH cells was calculated from

fields captured 4–8 min after CXCL12 stimulation.

Flow Cytometric Analysis

Murine single-cell suspensions were prepared from BM and spleen af-

ter erythrocyte depletion. Cells were stained with antibodies, as indi-

cated: B220-APC-Cy7, IgM-FITC or -APC, IgD-FITC, AA4.1-PE. Gates

in the BM and spleen were set according to previous reports (Cariappa

et al., 2007; Hardy et al., 1983; Li et al., 1993). Data were collected on

FACSCanto and analyzed by FlowJo software.

Statistical Analysis

Student’s t test was used for statistical analysis. The level of signifi-

cance is indicated by the p value. Data are presented as mean ± stan-

dard deviation (SD).

Supplemental Data

Four figures, four movies, and Experimental Procedures are available

at http://www.immunity.com/cgi/content/full/27/5/811/DC1/.

ACKNOWLEDGMENTS

We thank M. Armant, M. St. Andre, and M. Cohen for their help in FACS

analysis. We thank T. Bowman for preparing mouse BM sections. This

work was supported by grants from the National Institutes of Health

(HL56949 and HL074355).

Received: June 4, 2007

Revised: August 28, 2007

Accepted: September 25, 2007

Published online: November 26, 2007

Im

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