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Regulation of adipogenic differentiation by LAR tyrosine phosphatase in human mesenchymal stem cells and 3T3-L1 preadipocytes
Won-Kon Kim, Hyeyun Jung, Do-Hyung Kim, Eun-Young Kim, Jin-Woong Chung, Yee-Sook Cho, Sung-Goo Park, Byoung-Chul Park, Yong Ko, Kwang-Hee Bae, Sang-Chul Lee
  1. Fig. 1.

    LAR expression during adipogenic differentiation of human MSCs and 3T3-L1 preadipocytes. (A) Analysis of LAR mRNA expression during adipocyte differentiation of human MSCs by RT-PCR. (B) Analysis of LAR mRNA expression during adipocyte differentiation of human MSCs by real-time PCR. (C) Analysis of LAR expression during adipocyte differentiation of 3T3-L1 preadipocytes. LAR mRNA (upper) and protein (lower) expression levels decreased during adipocyte differentiation of 3T3-L1 preadipocytes. For RT-PCR analysis, total RNA was extracted on the indicated days of differentiation and used to synthesize cDNA for RT-PCR. Two primer sets were used to check for LAR expression, one for amplification of the mRNA region encoding the extracellular domain (Extra LAR) and another for amplification of the mRNA region encoding the cytoplasmic domain (Cyto LAR). The results obtained using the two sets of primers revealed similarly decreased LAR expression patterns during adipogenesis of 3T3-L1 cells. aP2 and PPAR-γ were used as positive controls for adipocyte differentiation and β-actin was used as a loading control. For western blot analysis, whole-cell lysates were extracted on the indicated days of differentiation, and subjected to analysis using antibodies against LAR and adipocyte-differentiation markers, such as aP2 and PPAR-γ. β-actin was used as a loading control. (D) Densitometric analysis of LAR protein expression during adipocyte differentiation of 3T3-L1 cells (means ± s.d.; n=3; *P<0.05).

  2. Fig. 2.

    LAR shRNA promotes 3T3-L1 adipogenic differentiation. (A) Direct monitoring of fluorescence protein expression using fluorescence microscopy after enrichment using a FACS sorter. 3T3-L1 cells were infected with retrovirus containing the vector alone (pSIREN-RetroQ-DsRed), scrambled insert or LAR shRNA (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 knockdown than the other constructs tested. Thus, this construct was used for the following phenotype 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 of lipid accumulation. (D) Quantification of Oil-red-O staining. Data represent the mean percentage levels ± s.d. compared with control vector (n=3; *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 or LAR 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. β-actin was used as a loading control. (F) The rescue experiment was performed by investigating 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. Six days later, cells were stained with Oil red-O. (G) Quantification of Oil-red-O staining. Data represent the mean percentage levels ± s.d. compared with control vector (n=3; *P<0.05).

  3. Fig. 3.

    Overexpression of LAR blocks adipocyte differentiation of 3T3-L1 cells. Cells were infected with retroviruses expressing FLAG-tagged cytoplasmic LAR or a LAR mutant (inactive mutant; catalytic domain Cys1522 replaced with Ser; D1-CS). Infected cells were selected by FACS sorting. (A) Expression of GFP was monitored directly by fluorescence microscopy. (B) Expression of cytoplasmic LAR was confirmed by western blot analysis. (C) Two days after post-confluence (day 0), cells overexpressing cytoplasmic or mutant LAR were induced to differentiate into mature adipocytes with MDI for 10 days. Cells were stained with Oil Red-O to visualize lipid droplets at 10 days post-induction. (D) Quantification of stained cells was performed using dye extraction buffer. Data represent the mean percentage levels ± s.d. compared with control vector (n=3; *P<0.05). (E) Whole-cell lysates were prepared on day 0 or 10 for western blot analysis of cytoplasmic LAR, aP2, PPAR-γ and β-actin.

  4. Fig. 4.

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

  5. Fig. 5.

    LAR dephosphorylates IR, IRS-1 and its downstream target Akt. (A,B) After obtaining confluent cultures of 3T3-L1 cells overexpressing the cytoLAR or LAR D1-CS mutant, cells were serum-starved for 12 hours and treated with 100 ng/ml insulin (A) or MDI (B) for the indicated times. (C,D) In the case of LAR-knockdown 3T3-L1 cells (by shLAR-II), cells were cultured to confluence, serum starved for 12 hours and treated with 100 ng/ml insulin (C) or MDI (D) for the indicated times. (A-D) Total cell lysates containing equal amounts of protein were immunoprecipitated with anti-IR-β and anti-IRS-1 antibodies, and western blot analysis was performed with anti-phosphotyrosine (4G10; anti-pY). The same membrane was reprobed and immunoblotted with anti-IR-β and -IRS-1 antibodies. Akt phosphorylation was measured with phospho-Akt antibody (antibody to phospho-Ser473) and the same membrane was immunoblotted with anti-Akt antibody.

  6. Fig. 6.

    Effects of LAR on differentiation into osteoblasts of MC3T3-E1 preosteoblast cells. (A) Analysis of LAR mRNA levels by RT-PCR during differentiation into osteoblasts. *P<0.05. (B) Direct monitoring of fluorescence protein expression using fluorescence microscopy after enrichment using a FACS sorter. MC3T3-E1 cells were stably transfected with empty vector or cytoplasmic LAR and vector only, scramble insert or LAR shRNA. Transfected cells were visualized by fluorescence microscopy. (C) Overexpression and knockdown of LAR were confirmed by western blot (left) and RT-PCR (right) analyses. (D) MC3T3-E1 LAR-knockdown cells (using the shLAR-II construct) were induced to undergo osteoblastic differentiation for 21 days, according to standard procedures. Cells were stained with Alizarin-Red-S to visualize the bone matrix. (E) Quantification of stained cells was performed. Data represent the mean percentage levels ± s.d. compared with control vector (n=3; *P<0.05). (F) MC3T3-E1 cells overexpressing cytoLAR or LAR D1-CS mutant were induced to differentiate into osteoblasts for 21 days, according to standard procedures. Cells were stained with Alizarin-Red-S to visualize the bone matrix. (G) Quantification of stained cells was performed using dye extraction buffer. Data represent the mean percentage levels ± s.d. compared with control vector (n=3; *P<0.05).