4160 Research Article

IntroductionAdipocytes are highly specialized cells that have an important rolein energy homeostasis by harboring energy reservoirs as lipiddroplets (Cornelius et al., 1994; Hwang et al., 1997). However, thesereservoirs are implicated in a host of major human health problems,because excessive or insufficient energy reserves result in metabolicdisorders, such as obesity and lipodystrophy (Garg, 2000; Otto andLane, 2005). Adipogenesis involves the formation of preadipocytesfrom mesenchymal progenitor cells and their subsequentdifferentiation into adipocytes (Gregoire et al., 1998; Rosen andSpiegelman, 2000). The cellular and molecular mechanisms ofadipocyte differentiation are regulated via activation of severaladipogenic intracellular signaling pathways.

Insulin signaling clearly has marked effects on adipogenesis. Themechanisms of insulin are mediated by a cascade of tyrosine-phosphorylation events initiated by the binding of insulin to itsreceptor (White and Kahn, 1994; White, 1997; White and Yenush,1998). Binding stimulates the kinase activity of the insulin receptor(IR) and the phosphorylation of IR substrates (IRSs), leading to theactivation of downstream signaling molecules, includingphosphoinositide 3-kinase (PI3K) and Akt (also known as PKB).Activated Akt regulates the activity of several downstream proteinsinvolved in gluconeogenesis, lipogenesis and adipogenesis (Whiteand Kahn, 1994; Wang and Sul, 1998; Czech and Corvera, 1999;Withers et al., 1999; Baudry et al., 2006; Rosen and MacDougald,

2006). Downstream components of the insulin signaling cascadeare crucial for adipogenesis. The loss of individual IRS proteins,including the combined deletion of IRS1 and IRS2, leads toinhibition of adipogenesis (Laustsen et al., 2002; Tseng et al., 2004;Rosen and MacDougald, 2006). Moreover, inhibition of PI3K andthe loss of Akt repress adipogenesis via regulation of adipogenicand anti-adipogenic transcription factors (Garofalo et al., 2003;Nakae et al., 2003; Wofrum et al., 2003; George et al., 2004;Menghini et al., 2005; Rosen and MacDougald, 2006).

Numerous growth-factor and -hormone receptors belong to thetyrosine-kinase-receptor family and undergo phosphorylation anddephosphorylation at tyrosine residues in a concerted manner inresponse to a stimulus in order to initiate the signaling cascade(Meng et al., 2004; Niu et al., 2007). Termination of insulin activityis also mediated by protein tyrosine phosphatases (PTPs), whichdephosphorylate and inactivate the IR and, subsequently, post-receptor substrates. PTPs constitute a large family of transmembraneor intracellular enzymes that function as either positive or negativeregulators of a number of signaling pathways (Goldstein, 1993;Denu et al., 1996; Tonks and Neel, 1996). Several PTPs, such asleukocyte common antigen related (LAR; also known as PTP-RF)phosphatase, PTP-a, PTP-1B and SHPTP2 (also known as Syp),are highly expressed in the major insulin-sensitive tissues, includingthe liver, skeletal muscle and adipose tissue. These enzymes areinvolved in insulin signaling, as established using a variety of

Mesenchymal stem cells (MSCs) are multipotent adult stem cellsthat can differentiate into a variety of mesodermal-lineage cells.MSCs have significant potential in tissue engineering andtherapeutic applications; however, the low differentiation andproliferation efficiencies of these cells in the laboratory arefundamental obstacles to their therapeutic use, mainly owingto the lack of information on the detailed signal-transductionmechanisms of differentiation into distinct lineages. With theaid of protein-tyrosine-phosphatase profiling studies, we showthat the expression of leukocyte common antigen related (LAR)tyrosine phosphatase is significantly decreased during the earlyadipogenic stages of MSCs. Knockdown of endogenous LARinduced a dramatic increase in adipogenic differentiation,whereas its overexpression led to decreased adipogenicdifferentiation in both 3T3-L1 preadipocytes and MSCs. LAR

reduces tyrosine phosphorylation of the insulin receptor, in turnleading to decreased phosphorylation of the adaptor proteinIRS-1 and its downstream molecule Akt (also known as PKB).We propose that LAR functions as a negative regulator ofadipogenesis. Furthermore, our data support the possibility thatLAR controls the balance between osteoblast and adipocytedifferentiation. Overall, our findings contribute to theclarification of the mechanisms underlying LAR activity in thedifferentiation of MSCs and suggest that LAR is a candidatetarget protein for the control of stem-cell differentiation.

Key words: Adipocyte, Adipogenesis, Mesenchymal stem cells,Osteoblast, Protein tyrosine phosphatase, Leukocyte common antigenrelated

Summary

Regulation of adipogenic differentiation by LARtyrosine phosphatase in human mesenchymal stemcells and 3T3-L1 preadipocytesWon-Kon Kim1,2, Hyeyun Jung1, Do-Hyung Kim1, Eun-Young Kim1, Jin-Woong Chung3, Yee-Sook Cho4,Sung-Goo Park1, Byoung-Chul Park1, Yong Ko2, Kwang-Hee Bae1,* and Sang-Chul Lee1,*1Medical Proteomics Research Center, and 4Development and Differentiation Research Center, KRIBB, Daejeon 305-806, Republic of Korea2Division of Life Science and Genetic Engineering, College of Life and Environmental Sciences, Korea University, Seoul 136-701, Republic ofKorea3Department of Biological Sciences, Dong-A University, Busan 604-714, Republic of Korea*Authors for correspondence (khbae@kribb.re.kr; lesach@kribb.re.kr)

Accepted 9 September 2009Journal of Cell Science 122, 4160-4167 Published by The Company of Biologists 2009doi:10.1242/jcs.053009

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experimental approaches (Drake and Posner, 1998; Goldstein et al.,1998).

LAR is a receptor-type PTP that has two tandem-repeatcytoplasmic phosphatase domains (D1 and D2), of which themembrane-proximal D1 domain possesses catalytic activity (Streuliet al., 1990; Nam et al., 1999). LAR is widely expressed in insulin-sensitive tissues. Increased association of LAR with the IR occursafter treatment with insulin (Ahmad et al., 1995; Ahmad andGoldstein, 1997). Several studies have reported that LAR isnegatively regulated during insulin signaling. Moreover,overexpression of LAR leads to suppression of insulin activity, andknockdown of LAR enhances IR phosphorylation and PI3K activityin hepatoma cells (Kulas et al., 1995; Kulas et al., 1996; Li et al.,1996). An in vivo study similarly reported that overexpression ofLAR in mouse skeletal muscle suppresses IR signaling (Zabolotnyet al., 2001). LAR also regulates insulin-like growth factor-1receptor signaling in vascular smooth-muscle cells (Niu et al., 2007).These reports strongly implicate LAR in insulin-mediated adipocytedifferentiation. There are no direct reports on the exact role of LARin adipogenesis, although earlier studies have demonstrated thatLAR expression is decreased during adipogenesis of humanmesenchymal stem cells (MSCs) (Song et al., 2006). The presentstudy was undertaken to determine the regulatory effects of LARon the differentiation of preadipocytes and human MSCs intoadipocytes.

ResultsExpression of LAR during the differentiation of MSCs and 3T3-L1 cells into adipocytesTo determine the roles of the PTP family during the early phase ofadipogenesis, we extensively assessed the expression changes ofall PTP members via reverse-transcriptase PCR (RT-PCR) analysis(data not shown). Interestingly, expression of LAR mRNA wasmarkedly decreased during adipocyte differentiation of humanMSCs (Fig. 1A). To obtain a detailed assessment of the LAR level,real-time PCR analysis was performed. LAR mRNA expression wasclearly decreased at the later as well as the early stages ofadipogenesis in MSCs (Fig. 1B). Next, we assessed LAR expressionin 3T3-L1 preadipocyte cell lines using both RT-PCR and westernblot analyses. As shown in Fig. 1C,D, LAR expression levels weresignificantly decreased during the differentiation of 3T3-L1preadipocytes into adipocytes. The protein stability of LAR seemedto be higher than that of LAR mRNA, but on the whole a correlationbetween them was observed. Levels of adipogenic markers, suchas aP2 (fatty-acid-binding protein; FABP) and PPAR-, wereincreased following hormonal stimulation. These results clearlyshow that LAR expression decreased during differentiation intoadipocytes in both MSCs and preadipocyte cells.

Depletion of LAR promotes the differentiation of 3T3-L1preadipocytes into mature adipocytesTo determine whether endogenous LAR influences differentiation intoadipocytes, we infected 3T3-L1 preadipocytes with a retrovirusexpressing LAR shRNA, scrambled insert or control vector only.Infected cells were isolated by FACS sorting and then cultured further.As shown in Fig. 2A (upper panels), cells infected with a retrovirusexpressing shRNA against LAR were successfully enriched.Knockdown of endogenous LAR expression was confirmed by RT-PCR and western blot analyses (Fig. 2B). Next, retrovirally transduced3T3-L1 preadipocytes were induced to differentiate into adipocytesfor 2 days in growth medium containing MDI (see Materials and

Methods), followed by further differentiation and maturation ingrowth medium supplemented with insulin for 6 days. In parallel,fat accumulation was visualized by staining lipid droplets with Oilred-O (Fig. 2C). Interestingly, depletion of LAR dramaticallyfacilitated the differentiation of these cells into mature adipocytescompared with control and scrambled retrovirus-infected cells (Fig.2D). Next, to exclude off-target effects of shRNA treatment, weattempted to rescue the effect on differentiation by re-introducing anshRNA-resistant cytoLAR or mutant LAR (D1-CS; activity-deadmutant) into the LAR-knockdown 3T3-L1 cells. Most of the re-infected cells were detected as both GFP- and RFP-positive byfluorescence microscopy (Fig. 2A, lower panels). Knockdown ofendogenous LAR and expression of shRNA-resistant cytoLAR ormutant LAR D1-CS were confirmed by RT-PCR analysis (Fig. 2E).Consistently, the knockdown of endogenous LAR mRNA levels wascontinuously maintained until 6 days after the adipogenicdifferentiation of 3T3-L1 cells (Fig. 2E). The re-introduction of wild-type cytoLAR into LAR-knockdown cells induced a full recovery ofthe differentiation rate. By contrast, the re-introduction of LAR D1-CS

Fig. 1. LAR expression during adipogenic differentiation of human MSCs and3T3-L1 preadipocytes. (A)Analysis of LAR mRNA expression duringadipocyte differentiation of human MSCs by RT-PCR. (B)Analysis of LARmRNA expression during adipocyte differentiation of human MSCs by real-time PCR. (C)Analysis of LAR expression during adipocyte differentiation of3T3-L1 preadipocytes. LAR mRNA (upper) and protein (lower) expressionlevels decreased during adipocyte differentiation of 3T3-L1 preadipocytes. ForRT-PCR analysis, total RNA was extracted on the indicated days ofdifferentiation and used to synthesize cDNA for RT-PCR. Two primer setswere used to check for LAR expression, one for amplification of the mRNAregion encoding the extracellular domain (Extra LAR) and another foramplification of the mRNA region encoding the cytoplasmic domain (CytoLAR). The results obtained using the two sets of primers revealed similarlydecreased LAR expression patterns during adipogenesis of 3T3-L1 cells. aP2and PPAR- were used as positive controls for adipocyte differentiation and -actin was used as a loading control. For western blot analysis, whole-celllysates were extracted on the indicated days of differentiation, and subjected toanalysis using antibodies against LAR and adipocyte-differentiation markers,such as aP2 and PPAR-. -actin was used as a loading control.(D)Densitometric analysis of LAR protein expression during adipocytedifferentiation of 3T3-L1 cells (means ± s.d.; n3; *P<0.05).

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showed no significant changes in the differentiation rate of the cells,indicating the crucial involvement of LAR phosphatase activity inadipogenesis (Fig. 2F,G).

Ectopic expression of LAR inhibits the differentiation of 3T3-L1preadipocytes into adipocytesDepletion of LAR induced a dramatic increase in the adipocytedifferentiation of 3T3-L1 cells, indicating that LAR phosphataseis involved in the negative regulation of adipocyte differentiation.

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To clarify the role of LAR in adipogenesis, we infected 3T3-L1cells with the cytoplasmic domain of LAR using a retroviralsystem (cytoLAR-IRES-GFP). LAR D1-CS and control vectorwere used as negative controls. Infected cells were isolated usinga FACS sorter. Most of the cells were GFP-positive byfluorescence microscopy (Fig. 3A). Overexpression of cytoplasmicand mutant LAR protein was verified by western blot analysisusing anti-FLAG and anti-LAR antibodies (Fig. 3B). Expressionof LAR protein was continuously detected during the late phasesof differentiation (Fig. 3E). Infected cells were induced todifferentiate into mature adipocytes, and lipid accumulation wasassessed by Oil-red-O staining 10 days after culturing withdifferentiation medium (Fig. 3C). Overexpression of wild-typeLAR resulted in a lower degree of lipid accumulation comparedwith control and LAR-mutant protein (Fig. 3C,D). This anti-adipogenic effect of LAR was also demonstrated in the adipogenicdifferentiation induced by treatment with insulin only (data notshown). Consistently, expression levels of adipogenic markers,such as aP2 and PPAR-, were reduced in mature adipocytes uponLAR overexpression (Fig. 3E).

Effects of LAR on adipocyte differentiation of human MSCsLAR and specific shRNA against LAR were stably expressed inhuman MSCs via retroviral infection to establish the importance ofLAR during the adipogenesis of human MSCs (Fig. 4A). LAR

Fig. 2. LAR shRNA promotes 3T3-L1 adipogenic differentiation. (A)Directmonitoring of fluorescence protein expression using fluorescence microscopyafter enrichment using a FACS sorter. 3T3-L1 cells were infected with retroviruscontaining the vector alone (pSIREN-RetroQ-DsRed), scrambled insert or LARshRNA (upper). In addition, retroviruses containing the vector alone (pRetroX-IRES-ZsGreen1), cytoLAR or LAR D1-CS mutant were used to re-infect LAR-knockdown cells (lower). Infected cells were then selected with a FACS sorter.(B)Knockdown of LAR was confirmed by western blot (upper) and RT-PCR(lower) analyses. As shown, shLAR-II provided more efficient LAR knockdownthan the other constructs tested. Thus, this construct was used for the followingphenotype experiments. (C)LAR-depleted cells (using the shLAR-II construct)were induced to differentiate into adipocytes by treatment with MDI medium.Six days later, the cells were stained with Oil red-O to visualize the degree oflipid accumulation. (D)Quantification of Oil-red-O staining. Data represent themean percentage levels ± s.d. compared with control vector (n3; *P<0.05).(E)Analysis of LAR mRNA levels during adipogenic differentiation of LAR-knockdown cells after re-introduction of the LAR-shRNA-resistant cytoLAR orLAR D1-CS mutant. Total RNA was extracted on day 0 or 6 for RT-PCR.Human LAR primer set (see Materials and Methods) was used for RT-PCR. -actin was used as a loading control. (F)The rescue experiment was performed byinvestigating the effect on differentiation of the re-introduction of an shRNA-resistant cytoLAR or LAR D1-CS mutant into LAR-knockdown 3T3-L1 cells.Cells were differentiated into adipocytes by the addition of MDI medium. Sixdays later, cells were stained with Oil red-O. (G)Quantification of Oil-red-Ostaining. Data represent the mean percentage levels ± s.d. compared with controlvector (n3; *P<0.05).

Fig. 3. Overexpression of LAR blocks adipocyte differentiation of 3T3-L1cells. Cells were infected with retroviruses expressing FLAG-taggedcytoplasmic LAR or a LAR mutant (inactive mutant; catalytic domainCys1522 replaced with Ser; D1-CS). Infected cells were selected by FACSsorting. (A)Expression of GFP was monitored directly by fluorescencemicroscopy. (B)Expression of cytoplasmic LAR was confirmed by westernblot analysis. (C)Two days after post-confluence (day 0), cells overexpressingcytoplasmic or mutant LAR were induced to differentiate into matureadipocytes with MDI for 10 days. Cells were stained with Oil Red-O tovisualize lipid droplets at 10 days post-induction. (D)Quantification of stainedcells was performed using dye extraction buffer. Data represent the meanpercentage levels ± s.d. compared with control vector (n3; *P<0.05).(E)Whole-cell lysates were prepared on day 0 or 10 for western blot analysisof cytoplasmic LAR, aP2, PPAR- and -actin.

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expression and knockdown were confirmed by western blot andRT-PCR analyses, respectively (Fig. 4B). Analogous to the dataobtained with 3T3-L1 preadipocytes, knockdown of LAR promotedadipocyte differentiation of human MSCs (Fig. 4C,D), whereas LARoverexpression suppressed adipogenic differentiation (Fig. 4E,F).

LAR affects insulin-mediated adipogenic signal transductionTo clarify the mechanism underlying LAR-induced regulation ofadipogenesis, we examined the phosphorylation states of IR, IRS-1,and its downstream target molecule Akt following treatment withinsulin or MDI. Consistent with a previous report (Li et al., 1996),tyrosine phosphorylation of IR in 3T3-L1 preadipocytes was rapidlyincreased in response to insulin or MDI. Notably, overexpression ofLAR led to the inhibition of insulin- and MDI-induced tyrosinephosphorylation of IR and IRS-1 (Fig. 5A,B). Consistently, thisdecrease in tyrosine phosphorylation resulted in reduced phospho-Akt levels in cells overexpressing LAR, compared with control cells(Fig. 5A,B). A marginal effect of activity-dead mutant LAR on thephosphorylation of IR, IRS-1 and Akt was detected, indicating thatthe phosphatase activity of LAR is directly involved in the regulationof adipogenesis. Next, we checked the phosphorylation states of IR,IRS-1 and its downstream target molecule Akt after knockdown ofLAR using an shRNA construct. LAR knockdown induced anincrease in the phosphorylation of IR and IRS-1, resulting inaugmented phospho-Akt levels (Fig. 5C,D). These results stronglysuggest that LAR functions as a negative regulator of adipogenesisby controlling the phosphorylation level of IR, IRS-1 and,consequently, Akt.

Effects of LAR on osteoblast differentiationTo establish whether LAR is involved in regulatingosteoblastogenesis, we first examined the change in the expressionlevel of LAR during osteoblastogenesis of MC3T3-E1 cells. Asexpected, the mRNA level of LAR was continuously increasedduring the differentiation of MC3T3-E1 cells into osteoblasts (Fig.6A). LAR shRNA or cytoplasmic LAR was stably introduced intoMC3T3-El preosteoblast cells using a retroviral system. Knockdownand overexpression of LAR were confirmed by fluorescencemicroscopy, western blot and RT-PCR analyses (Fig. 6B,C). Cellswere differentiated into osteoblasts by culturing them in osteogenicinduction medium for 21 days. Cells were then stained withAlizarin-Red-S solution to visually detect mineralization.Interestingly, cells depleted of LAR displayed a significantly lowerdegree of mineralization than control cells (Fig. 6D,E). By contrast,the degree of mineralization was significantly increased in cellsoverexpressing LAR, compared with control cells. LAR D1-CS

Fig. 4. Effects of LAR on adipogenic differentiation of human MSCs. MSCswere infected as described in Figs 2,3. (A)The infected cells were enriched byFACS sorting and confirmed by fluorescence microscopy. (B)The western blot(left) and RT-PCR (right) analyses were performed to confirm theoverexpression and knockdown of LAR, respectively. (C)LAR-depleted humanMSCs (using the shLAR-II construct) were induced into the adipogenic lineagefor 14 days, according to standard procedures, and the cells were then stainedwith Oil red-O to visualize the degree of lipid accumulation. (D)Quantificationof Oil-red-O staining. Data represent the mean percentage levels ± s.d. comparedwith control vector (n3; *P<0.05). (E)Human MSCs overexpressing thecytoLAR or LAR D1-CS mutant were induced to differentiate into adipocytesvia the adipogenic program for 14 days. Cells were stained with Oil Red-O tovisualize lipid droplets at 14 days post-induction. (F)Quantification of stainedcells was performed using dye extraction buffer. Data represent the meanpercentage levels ± s.d. compared with control vector (n3; *P<0.05).

Fig. 5. LAR dephosphorylates IR, IRS-1 andits downstream target Akt. (A,B)Afterobtaining confluent cultures of 3T3-L1 cellsoverexpressing the cytoLAR or LAR D1-CSmutant, cells were serum-starved for 12hours and treated with 100 ng/ml insulin (A)or MDI (B) for the indicated times. (C,D)Inthe case of LAR-knockdown 3T3-L1 cells(by shLAR-II), cells were cultured toconfluence, serum starved for 12 hours andtreated with 100 ng/ml insulin (C) or MDI(D) for the indicated times. (A-D)Total celllysates containing equal amounts of proteinwere immunoprecipitated with anti-IR- andanti-IRS-1 antibodies, and western blotanalysis was performed with anti-phosphotyrosine (4G10; anti-pY). The samemembrane was reprobed and immunoblottedwith anti-IR- and -IRS-1 antibodies. Aktphosphorylation was measured withphospho-Akt antibody (antibody to phospho-Ser473) and the same membrane wasimmunoblotted with anti-Akt antibody.

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displayed a mineralization rate similar to the control cells (Fig.6F,G). These results support the possibility that LAR is involvedin osteoblastogenesis as well as adipogenesis.

DiscussionTyrosine phosphorylation is one of the fundamental mechanismsunderlying the numerous important aspects of eukaryote physiology,including cell growth and differentiation. Adipocyte differentiationis regulated by tyrosine phosphorylation balanced by theantagonistic actions of protein tyrosine kinases (PTKs) and PTPs(Hunter, 1987; Hunter, 1998; Blume-Jensen and Hunter, 2001).

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Among the ~100 PTPs, several play important roles in insulinsignaling, including LAR. Insulin signaling promotes adipogenesis(Smith et al., 1988; Rosen and MacDougald, 2006); however,limited information is available on the relationship between LARand adipogenesis. In the present study, the effects of LAR onadipocyte differentiation and insulin signal transduction duringadipogenesis in 3T3-L1 preadipocytes and human MSCs wereinvestigated.

LAR mRNA and protein levels were markedly suppressed duringadipocyte differentiation, suggesting that LAR might be involvedin adipogenesis. The data are consistent with the previous findingthat LAR expression is decreased during differentiation intoadipocytes and increased during dedifferentiation (Zhou et al., 2005;Song et al., 2006). However, there is no direct evidence for themechanism of LAR action in adipogenesis, either in preadipocytesor MSCs. In our experiments, ectopic expression of the cytoplasmicregion of LAR in 3T3-L1 preadipocytes was associated withsignificant inhibition of adipogenesis (Fig. 3C,D), implying thatLAR is crucial for the adipocyte differentiation process. In addition,the overexpression of LAR, but not of the LAR D1-CS mutant,was associated with a marked decrease in PPAR- and aP2expression (Fig. 3E), indicating that the phosphatase functions viathe downregulation of these key adipogenic factors. The findingthat an increase in LAR levels resulted in the inhibition of theadipogenic differentiation of human MSCs (Fig. 4C) suggests thatLAR blocks adipogenesis in both committed preadipocytes andmulti-lineage stem cells. In addition, silencing of LAR in both 3T3-L1 cells and MSCs dramatically induced adipogenic differentiation.These results indicate that LAR has an anti-adipogenic effect inboth stem cells and committed cells.

LAR functions as a negative regulator of insulin signaling in cell-culture and in animal model studies (Kulas et al., 1995; Kulas etal., 1996; Li et al., 1996; Zabolotny et al., 2001). Because insulinplays an important role in adipogenesis, we propose that LARsuppresses adipogenesis by modulating insulin signaling throughits tyrosine-phosphatase activity. Consistent with previousobservations (Kulas et al., 1995; Kulas et al., 1996; Li et al., 1996),LAR markedly reduced IR tyrosine phosphorylation (possibly in adirect manner) following insulin or MDI treatment, which in turnled to decreased phosphorylation of the adaptor protein IRS-1 andits downstream signaling molecule Akt (Fig. 5A,B). Previousinvestigations reported that interference with the IRS-1- and Akt-associated signaling pathways represses adipogenesis (Laustsen etal., 2002; Garofalo et al., 2003; George et al., 2004; Tseng et al.,2004; Baudry et al., 2006). Our data clearly show that the reducedadipogenic differentiation of 3T3-L1 and human MSCs by LARoccurs via decreased activation of IRS-I and Akt. Whereascytoplasmic LAR was previously shown to have no effect onphosphorylation of components of the insulin signaling pathway inCHO cells (Zhang et al., 1996), the protein clearly affected theinsulin signaling pathway via dephosphorylation of IR under ourexperimental conditions. Furthermore, the marginal effect of theinactive LAR mutant strongly supports the importance ofphosphatase activity in controlling adipogenesis.

The relationship between bone and fat has been established atboth the developmental and physiological level. Duringdevelopment, mesenchymal precursor cells differentiate intoadipocytes, osteoblasts and other cell types in vitro, and areciprocal relationship exists such that increased osteoblastdifferentiation is associated with decreased adipocytedifferentiation and vice versa (Nuttall and Gimble, 2000; Ichida

Fig. 6. Effects of LAR on differentiation into osteoblasts of MC3T3-E1preosteoblast cells. (A)Analysis of LAR mRNA levels by RT-PCR duringdifferentiation into osteoblasts. *P<0.05. (B)Direct monitoring of fluorescenceprotein expression using fluorescence microscopy after enrichment using aFACS sorter. MC3T3-E1 cells were stably transfected with empty vector orcytoplasmic LAR and vector only, scramble insert or LAR shRNA. Transfectedcells were visualized by fluorescence microscopy. (C)Overexpression andknockdown of LAR were confirmed by western blot (left) and RT-PCR (right)analyses. (D)MC3T3-E1 LAR-knockdown cells (using the shLAR-IIconstruct) were induced to undergo osteoblastic differentiation for 21 days,according to standard procedures. Cells were stained with Alizarin-Red-S tovisualize the bone matrix. (E)Quantification of stained cells was performed.Data represent the mean percentage levels ± s.d. compared with control vector(n3; *P<0.05). (F)MC3T3-E1 cells overexpressing cytoLAR or LAR D1-CSmutant were induced to differentiate into osteoblasts for 21 days, according tostandard procedures. Cells were stained with Alizarin-Red-S to visualize thebone matrix. (G)Quantification of stained cells was performed using dyeextraction buffer. Data represent the mean percentage levels ± s.d. comparedwith control vector (n3; *P<0.05).

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et al., 2004). Interestingly, in contrast to adipogenic differentiation,osteoblast differentiation was augmented upon LARoverexpression and reduced following LAR depletion. Thisreciprocal effect of LAR on adipogenesis and osteoblastogenesisis consistent with previous findings that GILZ (glucocorticoid-induced leucine zipper) induces osteoblastogenesis and inhibitsadipogenesis in MSCs, and that TAZ (transcriptional co-activatorwith PDZ-binding motif) controls the balance of osteoblast andadipocyte differentiation (Hong et al., 2005; Zhang et al., 2008).In conjunction with earlier data, our results suggest that inhibitionof adipogenic differentiation by LAR occurs through theenhancement of osteogenic differentiation. Thus, LAR might actas a modulator of the balance between osteoblast and adipocytedifferentiation in MSCs. However, the reason for the marginaleffect of LAR on osteoblast differentiation, compared with thaton adipocyte differentiation, remains to be defined. Thus, extensivestudies to establish the mechanism underlying LAR involvementin osteoblastogenesis are warranted. In addition, LAR knockout(KO) mice were smaller in size than wild-type mice, were insulin-resistant and exhibited subtle alterations in neurogenesis, althoughthey have been reported to be, in general, normal and to possessno adipose-tissue or bone morphogenesis defects (Ren et al.,1998). Therefore, to elucidate the detailed functions of LAR inadipogenesis and osteoblastogenesis at an organism level, it isnecessary to conduct more detailed investigations of LAR KOmice.

Materials and MethodsCell cultureThe preadipocyte cell line 3T3-L1, derived from mouse embryo fibroblasts, wascultured in growth medium [high glucose Dulbecco’s modified Eagle’s medium(DMEM) containing 1% antibiotic-antimycotic solution and 10% bovine calf serum(BCS); Gibco-Invitrogen] at 37°C in a humidified atmosphere with 5% CO2.MC3T3-E1 preosteoblasts were purchased from ATCC and cultured in maintenancemedium [a-mineral essential medium (MEM) with 10% fetal bovine serum (FBS)and 1% antibiotic-antimycotic solution). Human MSCs were purchased from CambrexBio Science and maintained in MSC growth medium (MSCGM; Cambrex BioScience) at 37°C in a humidified atmosphere with 5% CO2.

Differentiation into adipocytes3T3-L1 cells were induced to differentiate into mature adipocytes, as describedpreviously (Kim et al., 2008; Jung et al., 2009). Briefly, at 2 days after confluence(day 0), cells were placed in differentiation medium composed of DMEM, 10% FBSand MDI, a differentiation cocktail of 0.5 mM isobutylmethylxanthine (IBMX), 1 Mdexamethasone and 10 g/ml insulin (Sigma), and then switched to maintenancemedium composed of DMEM, 10% FBS and 10 g/ml insulin. The medium wasreplenished every other day.

Adipocyte differentiation of human MSCs was induced as recommended by themanufacturer (Cambrex). In brief, after reaching confluence, the medium was switchedto adipogenic induction medium (MSCGM plus MDI and 200 M indomethacin;Cambrex) and the cells were cultured for 3 more days. The medium was switchedto adipogenic maintenance medium (MSCGM plus 10 g/ml insulin; Cambrex) for1-2 days. Three complete cycles of induction-maintenance medium were required tostimulate optimal adipogenic differentiation.

Differentiation into osteoblastsTo induce osteoblastogenesis, confluent MC3T3-E1 cells were switched tomaintenance medium (a-MEM with 10% FBS) supplemented with 50 g/ml ascorbicacid and 10 mM -glycerophosphate; the medium was changed every 2-3 days for21 days.

Osteoblast differentiation of human MSCs into mature osteoblasts was induced asdescribed previously (McBeath et al., 2004). Confluent MSCs were switched toosteogenic differentiation medium (50 M ascorbic acid, 10 mM -glycerophosphateand 100 nM dexamethasone; Sigma) and the medium was changed every 2-3 days.

Construction of retroviral vectors and transductionTo construct 3T3-L1 cells and MSCs stably expressing the FLAG-tagged cytoplasmicdomain of human LAR (spanning residues 1316-1897), a retrovirus-mediatedinfection system was used. For expression of cytoplasmic LAR, DNA encodingFLAG-tagged cytoplasmic LAR was inserted into the multi-cloning site of the

pRetroX-IRES-ZsGreen1 vector (Clontech). Retroviruses were subsequently producedby transiently co-transfecting GP2-293 cells with a retroviral vector and VSV-Gplasmid using Lipofectamine 2000 (Gibco-Invitrogen). At 48 hours after transfection,media containing retroviruses were collected, filtered with 0.45-m filters and usedto infect cells in the presence of polybrene (8 g/ml). Infected cells were selectedusing a FACSAria cell sorter (BD Bioscience) and further maintained in growthmedium. Ectopic expression of FLAG-tagged cytoplasmic LAR was confirmed bywestern blot analysis. As a negative control, a cytoplasmic LAR mutant (LAR D1-CS; inactive mutant; catalytic Cys1522 replaced with Ser) was constructed (Streuliet al., 1990; Nam et al., 1999).

RNA interferenceTo knock down LAR in 3T3-L1 cells, MC3T3-E1 cells and MSCs, the pSIREN-RetroQ-DsRed Express retroviral vector (Clontech) was employed. shRNAs weredesigned by selecting a target sequence specific for mouse and human LAR genes, asdescribed previously (Mander et al., 2005; Bernabeu et al., 2006). The following gene-specific sequences were used to successfully inhibit LAR expression in both humansand mice: shLAR-I: top, 5�-GATCCGGAATTACGTGGATGAAGAATTCAAGA-GATTCTTCATCCACGTAATTCTTTTTTG-3�, bottom, 5�-AATTCAAAAAA-GAATTACGTGGATGAAGAATCTCTTGAATTCTTCATCCACGTAATTCCG-3�(for mouse only); shLAR-II: top, 5�-GATCCGGGCCTACATAGCTACACAG -TTCAAGAGACTGTGTAGCTATGTAGGCCTTTTTTG-3�, bottom, 5�-AATTC -AAAAAAGGCCTACATAGCTACACAGTCTCTTGAACTGTGTAGCTATGTAG-GCCCG-3� (for both human and mouse). Each shRNA sequence was annealed andsubcloned according to the manufacturer’s guidelines, and non-targeting controlshRNA (scrambled; SCR) was obtained from Clontech.

shRNA-resistant cytoLAR and LAR D1-CS mutant were constructed by site-directed mutagenesis of the region that shLAR-II binds in order to degrade LARmRNA. Therefore, these constructs possess an altered mRNA sequence in the regioncorresponding to the shRNA sequence. The constructs were obtained by standardmethods using the primers 5�-GGCCTATATAGCCACACAGGGGCCTC-3� and 5�-TTCTGCTGTCTATAACCATCCAGGA-3�. Underlined regions indicate the mutatedsites (mutations did not induce a change in amino acid residues).

Immunoblot analysisCells were washed three times with ice-cold phosphate buffered saline (PBS)containing 1 mM sodium orthovanadate and harvested in ice-cold RIPA or NP-40lysis buffer containing a protease-inhibitor and phosphatase-inhibitor cocktail (Roche).Protein concentrations were measured with the BCA protein assay kit (Pierce). SDS-PAGE, western blot and densitometric analyses were performed using standardprotocols. The anti-LAR antibody is described in a previous report (Niu et al., 2007).Anti-aP2, anti-PPAR-, anti-phospho-Akt (cat. no. #9271S; antibody to phosphorylatedSer473), anti-Akt, anti-IR and anti-IRS-1 antibodies were purchased from CellSignaling. The anti-phosphotyrosine antibody (4G10) was obtained from Upstate,whereas anti-FLAG and anti--actin antibodies were from Sigma. The secondaryantibodies were purchased from Abcam, and membranes were visualized using theSuperSignal West Pico Chemiluminescent Substrate kit (Pierce).

ImmunoprecipitationFor immunoprecipitation, anti-IR or anti-IRS-1 antibodies (1:50-1:100 dilution) wereadded to lysates containing equal amounts of protein (200-300 g). The mixture wasincubated overnight at 4°C. Protein A/G plus agarose beads (Calbiochem) were added,followed by agitation for 1 hour at 4°C. Immunoprecipitates were recovered bycentrifugation at 2500 g, washed five times with NP-40 lysis buffer and analyzed asdescribed above.

RNA extraction and real-time PCRTotal RNA was extracted from cultured cells using RNeasy mini columns (QIAGEN),and first-strand complementary DNA (cDNA) was synthesized using 1 g of totalRNA as template, 500 ng of oligo (dT) and AccuPower RT Premix (Bioneer, Korea)in a total volume of 20 l, according to previously described methods (Jang et al.,2009). The primer sequences were as follows: mouse cytoplasmic LAR (forward, 5�-GTCAAGGCCTGTAACCCACT-3�, reverse, 5�-CCGTCTTCTCGTGCTTCATA-3�); mouse extracellular region of LAR (forward, 5�-CCACATCTACCACG-GAACTG-3�, reverse, 5�-GGCCCAAGAGTGTAAGGTGT-3�); human LAR(forward, 5�-ACCCGATGGCTGAGTACAAC-3�, reverse, 5�-GCACGGTAG-CACAGCTGATA-3�); mouse PPAR- (forward, 5�-GAGCACTTCACAAGAAAT-TACC-3�, reverse, 5�-GAACTCCATAGTGGAAGCCT-3�); mouse aP2 (forward,5�-GTGGGAACCTGGAAGCTTGTC-3�, reverse, 5�-CTTCACCTTCCTGTCG -TCTGC-3�). The targeted cDNA fragment of each differentiation-associated gene wasamplified by PCR using 5 l of the reverse transcription product, 10 pmol of eachprimer pair and the AccuPower PCR premix (Bioneer, Korea). PCR products wereseparated by electrophoresis in 2% agarose gels and visualized by staining withethidium bromide.

For the real-time PCR, SYYBR Premix Ex Tag (TaKaRa) was applied to detectthe LAR expression level using a Dice TP 800 Thermal Cycler (TaKaRa). Thefollowing primers were used for human LAR (forward, 5�-CCCTCATAAACCAAT-GTGGCAAG-3�, reverse, 5�-CCCTCAGCAAGCTGGGATAATAAG-3�) and -actin

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(forward, 5�-AGGCCCAGAGCAAGAGAGG-3�, reverse, 5�-TACATGGCTGGG -GTGTTGAA-3�).

Oil-red-O stainingLipid droplets in differentiating or mature adipocytes were stained using the Oil-red-O method, as described previously (Kim et al., 2008; Jung et al., 2009). In brief,cultured cells were washed twice with PBS, fixed for 30 minutes with 10% formalinand washed twice with distilled water prior to staining. Lipid droplets within the cellwere stained for 30 minutes using 0.3% filtered Oil-red-O solution in 60% isopropanol(Sigma). The cells were then washed twice with distilled water and micrographs wereobtained. Oil-red-O staining was then quantified, as described previously (Ramirez-Zacarias et al., 1992). After elution from fixed cells with absolute isopropanol, theextracted dye was measured with a GeneQuant 1300 spectrophotometer (GEHealthcare) at 510 nm.

Alizarin-Red-S stainingAlizarin-Red-S staining and mineral-content quantitation were performed followingearlier procedures with minor modifications (Stanford et al., 1995). Briefly, the cellswere washed with calcium and phosphate-free saline solution, and fixed with 10%formalin for 30 minutes. After washing with distilled water, the mineral content wasstained with 40 mM Alizarin-Red-S solution (pH 4.2, Sigma) at room temperaturefor 10 minutes. Cells were rinsed five times with distilled water and micrographswere obtained. Alizarin-Red-S was additionally quantified after elution from fixedcells with 10% (w/v) cetylpyridinium chloride (Sigma) and the absorbance wasmeasured at 570 nm using a GeneQuint1300 spectrophotometer (GE Healthcare).

Statistical analysisAll quantitative data were analyzed using an independent Student’s t-test andconsidered as significant at P<0.05.

We are grateful to Do Hee Lee, Sayeon Cho, Seung-Wook Chi,Sunghyun Kang and Chul Young Lee for careful reading of themanuscript. We also thank Hye Ryoung Choi for technical assistanceand helpful discussions. This work was supported by grants from KoreaResearch Council of Fundamental Science and Technology (KRCF),Korea Science and Engineering Foundation (KOSEF) and KRIBB.

ReferencesAhmad, F. and Goldstein, B. J. (1997). Functional association between the insulin receptor

and the transmembrane protein-tyrosine phosphatase LAR in intact cells. J. Biol. Chem.272, 448-457.

Ahmad, F., Considine, R. V. and Goldstein, B. J. (1995). Increased abundance of thereceptor-type protein-tyrosine phosphatase LAR accounts for the elevated insulinreceptor dephosphorylating activity in adipose tissue of obese human subjects. J. Clin.Invest. 95, 2806-2812.

Baudry, A., Yang, Z. Z. and Hemmings, B. A. (2006). PKBalpha is required for adiposedifferentiation of mouse embryonic fibroblasts. J. Cell Sci. 119, 889-897.

Bernabeu, R., Yang, T., Xie, Y., Mehta, B., Ma, S. Y. and Longo, F. M. (2006).Downregulation of the LAR protein tyrosine phosphatase receptor is associated withincreased dentate gyrus neurogenesis and an increased number of granule cell layerneurons. Mol. Cell Neurosci. 31, 723-738.

Blume-Jensen, P. and Hunter, T. (2001). Oncogenic kinase signalling. Nature 411, 355-365.

Cornelius, P., MacDougald, O. A. and Lane, M. D. (1994). Regulation of adipocytedevelopment. Annu. Rev. Nutr. 14, 99-129.

Czech, M. P. and Corvera, S. (1999). Signaling mechanisms that regulate glucose transport.J. Biol. Chem. 274, 1865-1868.

Denu, J. M., Stuckey, J. A., Saper, M. A. and Dixon, J. E. (1996). Form and functionin protein dephosphorylation. Cell 87, 361-364.

Drake, P. G. and Posner, B. I. (1998). Insulin receptor-associated protein tyrosinephosphatase(s): role in insulin action. Mol. Cell Biochem. 182, 79-89.

Garg, A. (2000). Lipodystrophies. Am. J. Med. 108, 143-152.Garofalo, R. S., Orena, S. J., Rafidi, K., Torchia, A. J., Stock, J. L., Hildebrandt, A.

L., Coskran, T., Black, S. C., Brees, D. J., Wicks, J. R., McNeish, J. D. and Coleman,K. G. (2003). Severe diabetes, age-dependent loss of adipose tissue, and mild growthdeficiency in mice lacking Akt2/PKB beta. J. Clin. Invest. 112, 197-208.

George, S., Rochford, J. J., Wolfrum, C., Gray, S. L., Schinner, S., Wilson, J. C., Soos,M. A., Murgatroyd, P. R., Williams, R. M., Acerini, C. L. et al. (2004). A familywith severe insulin resistance and diabetes due to a mutation in AKT2. Science 304,1325-1328.

Goldstein, B. J. (1993). Regulation of insulin receptor signaling by protein-tyrosinedephosphorylation. Receptor 3, 1-15.

Goldstein, B. J., Ahmad, F., Ding, W., Li, P. M. and Zhang, W. R. (1998). Regulationof the insulin signalling pathway by cellular protein-tyrosine phosphatases. Mol. CellBiochem. 182, 91-99.

Gregoire, F. M., Smas, C. M. and Sul, H. S. (1998). Understanding adipocytedifferentiation. Physiol. Rev. 78, 783-809.

Hong, J. H., Hwang, E. S., McManus, M. T., Amsterdam, A., Tian, Y., Kalmukova,R., Mueller, E., Benjamin, T., Spiegelman, B. M., Sharp, P. A., Hopkins, N. and

Journal of Cell Science 122 (22)

Yaffe, M. B. (2005). TAZ, a transcriptional modulator of mesenchymal stem celldifferentiation. Science 309, 1074-1078.

Hunter, T. (1987). A thousand and one protein kinases. Cell 50, 823-829.Hunter, T. (1998). The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine:

its role in cell growth and disease. Philos. Trans. R Soc. Lond. B Biol. Sci. 353, 583-605.Hwang, C. S., Loftus, T. M., Mandrup, S. and Lane, M. D. (1997). Adipocyte

differentiation and leptin expression. Annu. Rev. Cell Dev. Biol. 13, 231-259.Ichida, F., Nishimura, R., Hata, K., Matsubara, T., Ikeda, F., Hisada, K., Yatani, H.,

Cao, X., Komori, T., Yamaguchi, A. and Yoneda, T. (2004). Reciprocal roles of MSX2in regulation of osteoblast and adipocyte differentiation. J. Biol. Chem. 279, 34015-34022.

Jang, M., Park, B. C., Kang, S., Chi, S.-W., Cho, S., Chung, S. J., Lee, S. C., Bae, K.-H. and Park, S. G. (2009). Far upstream element-binding protein-1, a novel caspasesubstrate, acts as a cross-talker between apoptosis and the c-myc oncogene. Oncogene28, 1529-1536.

Jung, H., Kim, W. K., Kim, D. H., Cho, I. S., Kim, S. J., Park, S. G., Park, B. C., Lim,H. M., Bae, K.-H. and Lee, S. C. (2009). Involvement of PTP-RQ in differentiationduring adipogenesis of human mesenchymal stem cells. Biochem. Biophys. Res. Comm.383, 252-257.

Kim, W. K., Lee, C. Y., Kang, M. S., Kim, M. H., Ryu, Y. H., Bae, K.-H., Shin, S. J.,Lee, S. C. and Ko, Y. (2008). Effects of leptin on lipid metabolism and gene expressionof differentiation-associated growth factors and transcription factors during differentiationand maturation of 3T3-L1 preadipocytes. Endocr. J. 55, 827-837.

Kulas, D. T., Zhang, W. R., Goldstein, B. J., Furlanetto, R. W. and Mooney, R. A.(1995). Insulin receptor signaling is augmented by antisense inhibition of the proteintyrosine phosphatase LAR. J. Biol. Chem. 270, 2435-3438.

Kulas, D. T., Goldstein, B. J. and Mooney, R. A. (1996). The transmembrane protein-tyrosine phosphatase LAR modulates signaling by multiple receptor tyrosine kinases.J. Biol. Chem. 271, 748-754.

Laustsen, P. G., Michael, M. D., Crute, B. E., Cohen, S. E., Ueki, K., Kulkarni, R. N.,Keller, S. R., Lienhard, G. E. and Kahn, C. R. (2002). Lipoatrophic diabetes inIrs1(–/–)/Irs3(–/–) double knockout mice. Genes Dev. 16, 3213-3222.

Li, P. M., Zhang, W. R. and Goldstein, B. J. (1996). Suppression of insulin receptoractivation by overexpression of the protein-tyrosine phosphatase LAR in hepatoma cells.Cell Signal. 8, 467-473.

Mander, A., Hodgkinson, C. P. and Sale, G. J. (2005). Knock-down of LAR proteintyrosine phosphatase induces insulin resistance. FEBS Lett. 579, 3024-3028.

McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K. and Chen, C. S. (2004).Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev.Cell 6, 483-495.

Meng, T. C., Buckley, D. A., Galic, S., Tiganis, T. and Tonks, N. K. (2004). Regulationof insulin signaling through reversible oxidation of the protein-tyrosine phosphatasesTC45 and PTP1B. J. Biol. Chem. 279, 37716-37725.

Menghini, R., Marchetti, V., Cardellini, M., Hribal, M. L., Mauriello, A., Lauro, D.,Sbraccia, P., Lauro, R. and Federici, M. (2005). Phosphorylation of GATA2 by Aktincreases adipose tissue differentiation and reduces adipose tissue-related inflammation:a novel pathway linking obesity to atherosclerosis. Circulation 111, 1946-1953.

Nakae, J., Kitamura, T., Kitamura, Y., Biggs, W. H., 3rd, Arden, K. C. and Accili, D.(2003). The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Dev.Cell 4, 119-129.

Nam, H. J., Poy, F., Krueger, N. X., Saito, H. and Frederick, C. A. (1999). Crystalstructure of the tandem phosphatase domains of RPTP LAR. Cell 97, 449-457.

Niu, X. L., Li, J., Hakim, Z. S., Rojas, M., Runge, M. S. and Madamanchi, N. R.(2007). Leukocyte antigen-related deficiency enhances insulin-like growth factor-1signaling in vascular smooth muscle cells and promotes neointima formation in responseto vascular injury. J. Biol. Chem. 282, 19808-19819.

Nuttall, M. E. and Gimble, J. M. (2000). Is there a therapeutic opportunity to either preventor treat osteopenic disorders by inhibiting marrow adipogenesis? Bone 27, 177-184.

Otto, T. C. and Lane, M. D. (2005). Adipose development: from stem cell to adipocyte.Crit. Rev. Biochem. Mol. Biol. 40, 229-242.

Ramírez-Zacarías, J. L., Castro-Muñozledo, F. and Kuri-Harcuch, W. (1992).Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipidswith Oil red O. Histochemistry 97, 493-497.

Ren, J.-M., Li, P.-M., Zhang, W.-R., Sweet, L. J., Cline, G., Shulman, G. I., Livingston,J. N. and Goldstein, B. J. (1998). Transgenic mice deficient in the LAR protein-tyrosinephosphatase exhibit profound defects in glucose homeostasis. Diabetes 47, 493-497.

Rosen, E. D. and Spiegelman, B. M. (2000). Molecular regulation of adipogenesis. Annu.Rev. Cell Dev. Biol. 16, 145-171.

Rosen, E. D. and MacDougald, O. A. (2006). Adipocyte differentiation from the insideout. Nat. Rev. Mol. Cell Biol. 7, 885-896.

Smith, P. J., Wise, L. S., Berkowitz, R., Wan, C. and Rubin, C, S. (1998). Insulin-likegrowth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes. J.Biol. Chem. 263, 9402-9408.

Song, L., Webb, N. E., Song, Y. and Tuan, R. S. (2006). Identification and functionalanalysis of candidate genes regulating mesenchymal stem cell self-renewal andmultipotency. Stem Cells 24, 1707-1718.

Stanford, C. M., Jacobson, P. A., Eanes, E. D., Lembke, L. A. and Midura, R. J. (1995).Rapidly forming apatitic mineral in an osteoblastic cell line (UMR 106-01 BSP). J. Biol.Chem. 270, 9420-9428.

Streuli, M., Krueger, N. X., Thai, T., Tang, M. and Saito, H. (1990). Distinct functionalroles of the two intracellular phosphatase like domains of the receptor-linked proteintyrosine phosphatases LCA and LAR. EMBO J. 9, 2399-2407.

Tonks, N. K. and Neel, B. G. (1996). From form to function: signaling by protein tyrosinephosphatases. Cell 87, 365-368.

Jour

nal o

f Cel

l Sci

ence

4167Involvement of LAR during adipogenesis

Tseng, Y. H., Kriauciunas, K. M., Kokkotou, E. and Kahn, C. R. (2004). Differentialroles of insulin receptor substrates in brown adipocyte differentiation. Mol. Cell Biol.24, 1918-1929.

Wang, D. and Sul, H. S. (1998). Insulin stimulation of the fatty acid synthase promoteris mediated by the phosphatidylinositol 3-kinase pathway. Involvement of protein kinaseB/Akt. J. Biol. Chem. 273, 25420-25426.

White, M. F. (1997). The insulin signalling system and the IRS proteins. DiabetologiaSuppl. 2, S2-S17.

White, M. F. and Kahn, C. R. (1994). The insulin signaling system. J. Biol. Chem. 269,1-4.

White, M. F. and Yenush, L. (1998). The IRS-signaling system: a network of dockingproteins that mediate insulin and cytokine action. Curr. Top. Microbiol. Immunol. 228,179-208.

Withers, D. J., Burks, D. J., Towery, H. H., Altamuro, S. L., Flint, C. L. and White,M. F. (1999). Irs-2 coordinates Igf-1 receptor-mediated beta-cell development andperipheral insulin signalling. Nat. Genet. 23, 32-40.

Wolfrum, C., Shih, D. Q., Kuwajima, S., Norris, A. W., Kahn, C. R. and Stoffel, M.(2003). Role of Foxa-2 in adipocyte metabolism and differentiation. J. Clin. Invest. 112,345-356.

Zabolotny, J. M., Kim, Y. B., Peroni, O. D., Kim, J. K., Pani, M. A., Boss, O., Klaman,L. D., Kamatkar, S., Shulman, G. I., Kahn, B. B. and Neel, B. G. (2001).Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase inmuscle causes insulin resistance. Proc. Natl. Acad. Sci. 98, 5187-5192.

Zhang, W., Yang, N. and Shi, X. M. (2008). Regulation of mesenchymal stem cellosteogenic differentiation by glucocorticoid-induced leucine zipper (GILZ). J. Biol. Chem.283, 4723-4729.

Zhang, W. R., Li, P. M., Oswald, M. A. and Goldstein, B. J. (1996). Modulation ofinsulin signal transduction by ectopic overexpression of the receptor-type protein-tyrosinephosphatase LAR. Mol. Endocrinol. 10, 575-584.

Zhou, S., Lechpammer, S., Greenberger, J. S. and Glowacki, J. (2005). Hypoxiainhibition of adipocytogenesis in human bone marrow stromal cells requires transforminggrowth factor-beta/Smad3 signaling. J. Biol. Chem. 280, 22688-22696.

Jour

nal o

f Cel

l Sci

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