FMIP controls the adipocyte lineage commitment of C2C12 cells by downmodulation of C/EBPalpha

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Abstract

Fms interacting protein (FMIP) is a substrate for Fms tyrosine kinase, and a nuclear/cytoplasm shuttling protein with a leucine zipper. As the phosphorylation of FMIP is observed in insulin-stimulated preadipocytes, we examined the role of FMIP in adipocyte differentiation, using the mesenchymal multipotent stem cells, C2C12 cells, that can differentiate into adipocytes, muscle cells and osteoblasts. Ectopic expression of FMIP in C2C12 impairs the adipocyte differentiation induced by treatment with insulin, dexamethasone and 3-isobutyl-1-methylxanthine. These cells exhibit muscle phenotype with multinuclear morphology. Furthermore, knockdown of endogenous FMIP expression by small interfering RNA improves adipocytic lineage commitment of C2C12 cells, while impairing muscle differentiation. Upon stimulation with insulin, CCAAT/enhancer binding protein (C/EBP)beta, but not C/EBPalpha, is upregulated in cells expressing ectopic FMIP, whereas in FMIP knockdown cells, C/EBPalpha is constitutively expressed. Ectopic expression of C/EBPalpha counteracts the effects of FMIP, whereas C/EBPalpha knockdown partially mimics the effects of FMIP in this system. Northern blot analysis and reverse transcriptase–polymerase chain reaction study reveal that ectopic FMIP-expressing cells do not contain the polyadenylated C/EBPalpha mRNA, but contain the C/EBPalpha pre-mRNA, suggesting that FMIP plays a role in RNA processing and/or export. Indeed, a member of the THO complex that plays a role in mRNA export, THOC1, is co-precipitated with FMIP. The data we have acquired on FMIP suggest that it is a target for tyrosine kinase receptors that potentiate mRNA export.

Introduction

Fms interacting protein (FMIP) is a substrate, as well as a binding partner, of Fms tyrosine kinase (Tamura et al., 1999). FMIP is a ubiquitous nuclear/cytoplasm shuttling protein with a leucine zipper (Mancini et al., 2004). The overexpression of FMIP in myeloid progenitor cells alters the macrophage colony stimulating factor (M-CSF)-mediated macrophage differentiation. These cells differentiate into the granulocytic lineage rather than into the macrophage lineage (Tamura et al., 1999). Furthermore, it has been shown that FMIP is one of the major molecules phosphorylated via the insulin-mediated signaling pathway in a preadipocyte cell line, 3T3-L1 cells (Gridley et al., 2005), suggesting that FMIP may play a role in adipocyte differentiation.

The multipotent C2C12 mesenchymal progenitor cells are capable of differentiating into a number of cell types, including adipocytes, muscle cells and osteoblasts. Upon stimulation with insulin, dexamethasone and 3-isobutyl-1-methylxanthine (IBMX), C2C12 cells are differentiated into adipocytes. A number of transcription factors are known to be activated during adipocyte differentiation. A prominent transcription factor family is the CCAAT/enhancer binding protein (C/EBP) family (Otto and Lane, 2005). C/EBPalpha and C/EBPbeta are members of the family of six functionally and structurally related basic leucine zipper DNA binding proteins. The genes regulated by the C/EBP proteins are diverse and are involved in a variety of different cell processes including hematopoiesis, adipogenesis and morphogenesis (Johnson, 2005).

In this paper, we demonstrate that ectopic expression of FMIP in mesenchymal progenitor cell line C2C12 cells impaired adipocyte differentiation. Cells therefore become muscle cells rather than adipocytes. In these cells, C/EBPalpha is not upregulated upon stimulation with the adipocyte differentiation medium. Approximately a 70% reduction of endogenous FMIP expression leads to C/EBPalpha upregulation followed by adipocyte differentiation even in the absence of insulin. However, these cells failed to differentiate into muscle cells. Furthermore, we have shown that the modulation of C/EBPalpha expression by FMIP takes place at the mRNA processing and/or nuclear export level. Indeed FMIP co-precipitates THOC1, which is one of the components of the mRNA export complex. Thus, we have shown that a nuclear/cytosolic protein that is a target for receptor-mediated phosphorylation is also involved in mRNA export.

Results

FMIP is expressed in C2C12 cells and is upregulated during muscle differentiation

It has been previously shown that using AKT substrate motif (PAS) antibody (Kane et al., 2002), FMIP in 3T3-L1 cells is highly phosphorylated within 10 min after insulin stimulation (Gridley et al., 2005). To examine whether FMIP plays a role in insulin-induced adipocyte differentiation, we first examined FMIP expression in the multipotent mesenchymal stem cell line C2C12. FMIP is clearly detected by immunoblotting using a monoclonal antibody against FMIP. The level of endogenous FMIP is not altered during adipocyte differentiation induced by treatment with dexamethasone, IBMX and insulin. The expression of FMIP is upregulated approximately twofold after treatment with muscle differentiation medium containing 2% fetal calf serum (FCS) and 8% horse serum (HS) (Figure 1a). In agreement with these data, we have shown previously that FMIP is expressed in the skeletal muscle and heart at high levels (Tamura et al., 1999). In addition, both C/EBPalpha and C/EBPbeta are upregulated in medium that promotes adipocyte differentiation, but not in conditions that promote muscle differentiation (Figure 1a). The robust nature of these differentiation assays was confirmed using standard tests: accumulation of Oil Red-positive lipid droplets was observed in C2C12 cells incubated with adipocyte differentiation medium; cells were fused in muscle differentiation medium within 8 days (Figure 1b).

Figure 1
figure1

FMIP is upregulated in C2C12 cells during muscle differentiation. C2C12 cells were grown in adipocyte differentiation medium (Insulin, Dex, IBMX or adipocyte) or muscle differentiation medium (8% HS, 2% FCS or muscle) for 8 days. (a) The same protein amount of each cell lysate was applied for Western blot using FMIP-, C/EBPbeta-, C/EBPalpha-, actin- and Erk1/2-specific antibodies. (b) Cells were fixed with 4% paraformaldehyde (PFA) and stained by ORO and hematoxylin or Giemsa.

Ectopic expression of FMIP in C2C12 cells impairs the adipocyte differentiation

To examine the role of FMIP in adipocyte/muscle differentiation, we first expressed myc-tagged FMIP ectopically. After G418 selection, we isolated 20 individual clones and five of them expressed detectable amounts of myc-tagged FMIP. The level of expression of all clones was about 1.5- to twofold more than endogenous FMIP measured by LAS3000 using the program Aida (Fuji Film, Kanagawa, Japan) (Figure 2a). All clones showed threefold increase of cell growth and differentiated myoblastic morphology: cells grew in the same direction and lost actin cables (Figure 2b); however, the early muscle marker, myogenin, was not detectable in the ectopic FMIP-expressing cells. None of the empty vector transfected C2C12 cells showed changed morphology. In the presence of adipocyte-differentiation medium for 8 days, the difference was more obvious. The ectopic FMIP expressing cells did not differentiate into adipocytes in this period. The morphology of two representative clones, FMIP#7 and FMIP#10, is shown in Figure 2c. These cells began to fuse in the presence of adipocyte differentiation medium and fewer cells were stained by Oil Red O (ORO). In the empty vector-transfected cell line, V#2, the accumulation of Oil Red-positive lipid droplets was observed. To confirm these data, we utilized the mouse preadipocyte cell line 3T3-L1 cells. The ectopic expression of FMIP in 3T3-L1 cells also impaired adipocyte differentiation (data not shown). In the muscle differentiation medium for 8 days, all C2C12 transfectants underwent alignment and fusion to multinucleated tubes (Figure 2c).

Figure 2
figure2

Ectopic expression of FMIP in C2C12 cells blocked adipocyte differentiation. Myc/His-tagged FMIP cDNA was transfected into C2C12 cells and after G418 selection, colonies were isolated. (a) Cell lysates of two cloned cell lines, #7 and #10, were applied for Myc-, FMIP- and actin-specific immonoblot. (b) Cells were fixed PFA and stained with Giemsa or tetramethylrhodamine B isothiocyanate-conjugated phalliodine (actin). As control, we utilized the empty vector-transfected C2C12 cells (vector#2). (c) The same cell lines were incubated in adipocyte differentiation medium or muscle differentiation medium for 8 days. Cells were fixed with 4% PFA and stained by ORO and hematoxylin or Giemsa.

Downregulation of endogenous FMIP expression impairs muscle differentiation and accelerates adipocyte differentiation of C2C12 cells

We further examined the role of FMIP by knocking down the endogenous FMIP applying short hairpin RNA (shRNA) interference technique using a lentiviral vector (Scherr et al., 2003). After infection of C2C12 cells with the lentivirus carrying shRNA to knock down FMIP expression, we obtained 12 clones by the limiting dilution and all of them were green fluorescent protein positive. Approximately a 70% reduction of endogenous FMIP expression was observed in clone 3#1 (Figure 3a). As expected, control cells infected with a lentivirus carrying shRNA against Gal4 (Scherr et al., 2003) responded to the differentiation medium by accumulation of Oil Red-positive lipid droplets into an enlarged cytoplasm (Figure 3b). Inhibition of FMIP expression improved adipocytic lineage commitment in C2C12 cells. Both the silenced clones show adipocytic morphology, monitored as increase of intracellular lipid storage under normal growth conditions. Moreover, fat loading was drastically upregulated upon stimulation in comparison to control cells within the 8 days of treatment (Figure 3b). In contrast to ectopic FMIP-expressing cells, FMIP knockdown cells did not undergo alignment, elongation and fusion to multinucleated tubes under myogenic differentiation medium. In addition, ORO staining of these cells demonstrates that FMIP downregulation resulted in acceleration of lipid inclusion in myocytes.

Figure 3
figure3

Knockdown of FMIP expression blocks muscle differentiation and rather accelerates adipocyte differentiation. C2C12 cells were infected with lentivirus carrying shGal4 (Scherr et al., 2003) or shFMIP-RNA and isolated cell lines. siFMIP#3–1 cells express only 30% of endogenous FMIP compared to Gal4#3 cells. (a) The same protein amount of cell lysates was applied onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis at three different concentrations followed by FMIP- and Erk1/2-specific Western blot. The intensity of each band was measured by LAS3000 using the program ‘Aida’. (b) The same cell lines were grown in adipocyte differentiation medium or in muscle differentiation medium for 8 days. Cells were fixed and stained by ORO and hematoxylin.

FMIP influences the expression of C/EBPalpha gene during adipocyte differentiation

The next issue considered was how FMIP exerts its effects on adipocyte differentiation. A number of proteins play a key role in adipocyte differentiation of C2C12 cells. As FMIP influences myeloid differentiation as well, we examined the common key factors in both differentiation systems. One candidate is a protein belonging to the C/EBP family (Cao et al., 1991; Wang et al., 1995; Tenen et al., 1997; Lane et al., 1999), as it has been demonstrated that timely expression of C/EBPalpha and C/EBPbeta is essential for adipocyte differentiation (Fux et al., 2004; Mori et al., 2005). Therefore, we examined the expression of C/EBPalpha and C/EBPbeta during differentiation in FMIP-overexpressing and knockdown cells. Although C/EBPbeta was upregulated within 2 days after treatment with adipocyte differentiation medium in all cell lines, C/EBPalpha was not expressed in ectopic FMIP-expressing cell lines (Figure 4a). In control cells, V#2, C/EBPalpha expression was clearly upregulated within 4 days after treatment. Park et al. (2004) demonstrated that phosphorylation of C/EBPbeta is required for C/EBPalpha expression. As shown in Figure 4a, the phosphorylation of C/EBPbeta is identical in ectopic FMIP-expressing cells and in the vector-transfected control cells, V#2. To examine whether FMIP downregulates C/EBPalpha expression at the post-translational level or at the mRNA level, we performed C/EBPalpha-specific Northern blots using ectopic FMIP-expressing and control vector C2C12 (V#2) cells. The total RNA was analysed by C/EBPalpha-specific and actin-specific Northern blotting. As shown in Figure 4b, all cells express the C/EBPalpha gene at approximately the same levels. We next examined the amount of polyadenylated C/EBPalpha mRNA by reverese transcriptase–polymerase chain reaction (RT–PCR). The total RNAs were reverse transcribed by oligo-dT and then C/EBPalpha-specific PCR was performed. The C/EBPalpha transcript was detected in V#2 cells, but not in ectopic FMIP-expressing cells (Figure 4c). On the other hand, in agreement with data obtained from morphological experiments (Figure 3), in FMIP knockdown cells, C/EBPalpha is expressed even in the presence of muscle differentiation medium (Figure 4c), suggesting that FMIP downmodulates polyadenylated C/EBPalpha mRNA expression under these conditions.

Figure 4
figure4

Ectopic expression of FMIP impaired the upregulation of C/EBPalpha during adipocyte differentiation. C2C12 cells that expressed FMIP ectopically (FMIP#7) and vector-transfected control cells (V#2) were grown in the presence of adipocyte differentiation medium for 8 days. (a) Cell lysates were supplied for Western blotting using FMIP-, C/EBPbeta-, phospho-C/EBPbeta- or C/EBPalpha-specific antibodies. As control, actin or Erk1/2 antibodies were used. (b and c) Ectopic FMIP-expressing cells (FMIP#7), empty vector-expressing cells (V#2) and FMIP knockdown cells (siFmip3#1) were incubated with adipocyte differentiation medium (Adi) or muscle differentiation medium (Mus) for 2 and 8 days. Isolated total RNAs from cells were applied for Northern blotting using C/EBPalpha and actin genes as probe (b). Same materials were reverse transcribed using oligo-dT and then applied for PCR using C/EBPalpha-specific primers as described in Materials and methods (Shi et al., 2003). As control, RT–PCR without template (c) was performed. PCR products were analysed by agarose gel electrophoresis followed by ethidium bromide staining. As control, actin primers were used (c).

C/EBPalpha counteracts the effect of FMIP on adipocyte/myocyte differentiation

Recently, a mammalian homologue of THOC5 in Drosophila melanogaster which is a member of the nuclear export complex (Rehwinkel et al., 2004), was identified as fSAP79 (Masuda et al., 2005). The amino-acid sequence of fSAP79 is identical to FMIP. To confirm that FMIP is a member of THO complex, we immunoprecipitated FMIP using monoclonal antibody against FMIP or Myc and then performed THOC1-specific Western blotting. Using this approach, it was demonstrated that FMIP forms a complex with THOC1 in C2C12 cells (data not shown), suggesting that FMIP/THOC5 may be a novel signaling molecule linking receptor tyrosine kinase activation to the regulation of nuclear export of mRNA. However, the role of the THO complex in the mammalian system is not well studied. In the Drosophila system, it has been shown that THO is required for efficient export of only a few mRNA (Rehwinkel et al., 2004). Nevertheless, this raises the question whether the FMIP-induced phenotype is owing to C/EBPalpha protein alone or if additional proteins are involved. We first expressed, therefore, C/EBPalpha in FMIP#7 and control V#2 cells ectopically and then incubated in adipocyte differentiation medium for 5 days. After fixation, C/EBPalpha was visualized using C/EBPalpha antibody and anti fluorescein isothiocyanate (FITC)-conjugated anti rabbit IgG (C/EBPalpha). Then, cells were stained by ORO. As shown in Figure 5a, more than 80% of C/EBPalpha-positive FMIP#7 cells contain lipid droplets, indicating that C/EBPalpha alone reverses the phenotype induced by FMIP. To examine whether C/EBPalpha downregulation in C2C12 cells causes muscle differentiation in the presence of adipocyte differentiation medium, we utilized lentivirus containing shRNA of C/EBPalpha. Cells infected by virus containing shC/EBPalpha express similar amounts of basal C/EBPalpha, however, upon stimulation with adipocyte differentiation medium, C/EBPalpha is upregulated about 10-fold in control virus-infected cells (siGAL4) but not in siC/EBPalpha cells (Figure 5b). Interestingly, C/EBPalpha downregulated cells grow about twofold faster than control virus carrying shGAL4-infected cells (Figure 5c), but do not show the differentiated myoblastic morphology in normal medium. In the presence of adipocyte differentiation medium, C/EBPalpha downregulated cells contain lipid droplets to a lesser extent (Figure 5d), but differentiate into myoblastic cells in the presence of muscle differentiation medium (data not shown), indicating that C/EBPalpha is a key molecule for trans-differentiation of C2C12 cells.

Figure 5
figure5

Ectopic expression of C/EBPalpha counteracts the effects of FMIP, whereas C/EBPalpha knockdown mimics the effect of FMIP. (a) Ectopic C/EBPalpha expression counteracts the inhibition of C2C12 cells adipogenesis by FMIP: Ectopic FMIP-expressing cells (FMIP#7) and empty vector transfectants (V#2) were transfected with C/EBPalpha cDNA and incubated with the adipocyte differentiation medium for 5 days. After fixation, C/EBPalpha was visualized using C/EBPalpha antibody and anti-FITC-conjugated anti-rabbit IgG (C/EBPalpha). Then, cells were stained by ORO. Arrows show C/EBPalpha-positive cells and the same cells contain ORO-positive droplets. (b–d) C/EBPalpha siRNA downmodulates adipocyte differentiation of C2C12 cells: C2C12 cells were infected with lentivirus carrying C/EBPalpha shRNA (siC/EBPα). As control, cells infected with control virus carrying Gal4 shRNA (siGAL4) were applied. Western blotting analysis using C/EBPalpha-, FMIP- or GAPDH-specific antibody in the absence (Day 0) or presence of adipocyte differentiation medium for 2 and 4 days (b). Aliquots of 1 × 104 cells of siGAL4 and siC/EBPalpha cells were grown in medium containing 10% FCS for 2 days. Cells were stained with Trypan blue and living cells were counted. Cell numbers are presented as the mean±s.e. for three independent experiments performed in triplicate (c). The same cell lines were incubated in adipocyte differentiation medium for 4 days. Cells were fixed with 4% PFA and stained by ORO and hematoxylin (d).

Discussion

We recently described a protein that binds to activated c-Fms, namely FMIP. FMIP is widely expressed; however, it is especially strongly expressed in muscle cells, liver and bipotent neutrophil/macrophage progenitor cells (Tamura et al., 1999). The results reported in this paper demonstrate the following (1) FMIP is highly expressed in the mesenchymal progenitor C2C12 cell line that can differentiate into adipocyte, chondrocyte, myoblast and osteoblast lineage. (2) Ectopic expression of FMIP in C2C12 cells abrogates insulin-mediated adipocyte differentiation as well as upregulation of C/EBPalpha at the mRNA processing or/and its export level, causing the cells to take on the myoblast like phenotype. (3) Knockdown of FMIP gene in these cells disables myoblast differentiation, whereby C/EBPalpha is simultaneously upregulated. In these cells, C/EBPalpha was detected even in the presence of muscle differentiation medium. (4) Ectopic expression of C/EBPalpha counteracts the FMIP-induced differentiation phenotype and siRNA inhibition of C/EBPalpha partially mimics the effect of FMIP. It has been clearly demonstrated that C/EBPalpha is a key player in adipocyte differentiation (Cao et al., 1991; Lin and Lane, 1994; Hu et al., 1995). For example, fibroblasts differentiated into adipocytes following ectopic expression of C/EBPalpha (Freytag et al., 1994; Park et al., 2004). It has also been reported that the C/EBPalpha-mediated myoblast to adipocyte transition of C2C12 takes place in the absence of adipocyte differentiation medium (Fux et al., 2004). C/EBPbeta and C/EBPalpha are also key molecules for myeloid development (Friedman, 2002) or hepatocyte differentiation (Flodby et al., 1996). C/EBPalpha −/− mice lack neutrophils and eosinophils, but retain monocytes, lymphocytes, erythroid cells and immature myeloblasts (Zhang et al., 1997). We have previously demonstrated that ectopic expression of FMIP in the myeloid progenitor cell line, FDC-P1Mac11, causes granulocyte differentiation upon stimulation with M-CSF. In the FMIP-overexpressing myeloid cell line, C/EBPbeta is somewhat upregulated (AD Whetton and T Tamura, unpublished observation); however, the molecular mechanism of the upregulation is still under study. In these cells, C/EBPalpha is not expressed at a detectable level. The difference in the role of C/EBPalpha between the adipocyte system and granulocyte system is not clear. Interestingly, C/EBPalpha is required for adipocyte differentiation, whereas in the granulocyte differentiation system C/EBPalpha can be replaced by C/EBPbeta (Popernack et al., 2001; Jones et al., 2002; Wang and Friedman 2002). However, only a low level of C/EBPalpha is sufficient for granulocyte differentiation. Indeed, siRNA inhibition of C/EBPalpha mRNA did not influence the granulocytic colony formation of CD34+ primitive hematopoietic cells (M Scherr and M Eder, unpublished data). On the other hand, C/EBPalpha is absolutely responsible for termination of mitotic clonal expansion in adipocyte differentiation (Zhang et al., 2004), whereas C/EBPbeta has been shown to be promitotic in this system (Tang et al., 2003). As stated above, THOC5 is a member of the nuclear export complex in D. melanogaster (Rehwinkel et al., 2004), and is the orthologue of FMIP. Indeed THOC1, a member of THO complex, was co-immunoprecipitated with FMIP, and the amount of THOC1/FMIP complex was increased in the presence of adipocyte differentiation medium (A Mancini and O El Bounkari, unpublished data). At present, the role of the THO complex in the mammalian system is not clear. In the Drosophila system, it has been shown that THO complex is required for efficient export of only a few mRNAs (Rehwinkel et al., 2004). We demonstrate here that FMIP/THOC5 suppresses the mRNA processing/export of C/EBPalpha. However, the role of FMIP/THOC5 in the THO complex at the molecular level is still unclear and requires further study. Our preliminary data obtained from the DNA array technique reveal that about 7% of genes were influenced by FMIP in C2C12 cells (A Mancini, unpublished data). Nevertheless, as FMIP is highly phosphorylated upon stimulation with several tyrosine kinase receptors, such as receptor for M-CSF (Tamura et al., 1999, Mancini et al., 2004), insulin (Gridley et al., 2005) and nerve growth factor (El Bounkari and Tamura, unpublished data), FMIP/THOC5 may be the novel connection between tyrosine kinase signaling and the mRNA export process.

Materials and methods

Cells and antibodies

Mouse C2C12, HEK293T and 3T3 L1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) FCS. For adipocyte differentiation, confluent cells were incubated with dextamethasone and IBMX for 2 days and then incubated in the medium containing insulin (Sigma, München, Germany) for 6 days. For muscle differentiation, confluent cells were grown in medium containing 8% HS and 2% FCS for 8 days. Monoclonal antibodies against Myc epitope (9E10) and polyclonal antibodies against C/EBPalpha, C/EBPbeta and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Erk1/2 and phospho-C/EBPbeta were from Cell Signaling Technology (Beverly, MA, USA) and monoclonal antibody against THOC1 (p84 N5) was from Gene Tex Inc. (San Antonio, TX, USA).

Production and validation of an FMIP monoclonal antibody

The 3′ 2.25 kb EcoRV–EcoRI human FMIP cDNA fragment was subcloned into the vector pGEX-3X. This construct was used to express the C-terminus of the human FMIP protein as a glutathione-S-transferase (GST) fusion protein in Escherichia coli strain BL21. The purified recombinant protein was used to raise the FMIP monoclonal antibody F6D/11, using previously described techniques (Mason et al., 1983). The antibody specifically recognized the GST-FMIP recombinant protein and not GST alone by enzyme-linked immunosorbent assay. The 3′ 2.5 kb BamHI–EcoRI human FMIP cDNA fragment was cloned into the vector pcDNA4.1 HisMax. COS cells were transfected with this epitope-tagged FMIP expression plasmid using the Fugene reagent, according to the manufacturer's instructions (Roche). The antibody recognized the FMIP protein by immunohistochemical labeling of cytospin preparations (detection using the DAKO EnvisionTM system) and by Western blotting of cell lysates, prepared from the FMIP COS cell transfectants. The antibody also recognized the FMIP protein in paraffin-embedded FMIP transfectants by immunohistochemistry, indicating its utility for detecting the FMIP protein in routinely fixed human tissues.

Plasmid constructions and establishment of cell lines

To generate Myc/His-tagged FMIP, the EcoRI–DraI (1–2047) fragment of hFMIP cDNA (Tamura et al., 1999) was cloned into pcDNA3.1/Myc-His (Invitrogen, Carlsbad, NM, USA). DNA transfection was performed with the Polyfect™ reagent as described by the manufacturer (Qiagen, Hilden, Germany). Twenty different neomycin-resistant cell clones were obtained by growth in G418-containing selection medium. For generating shRNA, DNA oligonucleotides corresponding to position 366–384 of the murine FMIP gene (GenBank accession no. NM 172438) and to position 822–840 of the sequence of the murine CEBPalpha gene (GenBank accession no. NM_00767) were subjected to BLAST homology search, and thereafter chemically synthesized including overhang sequences from a 5′ BglII and a 3′ SalI restriction site for cloning purposes (BioSpring, Frankfurt, Germany). The numbering of the first nucleotide of the shRNA refers to the ATG start codon. The oligonucleotide sequences were as follows:

FPFMIP::

5′-IndexTermGATCCCCTTAGCCCACATCAGATTAATTCAAGAGATTAATCTGATGTGGGCTAATTTTTTGGAAG-3′

RPFMIP::

5′-IndexTermTCGACTTCCAAAAAATTAGCCCACATCAGATTAATCTCTTGAATTAATCTGATGTGGGCTAAGGG-3′

FPC/EBPalpha::

5′-IndexTermGATCCCCAGCCAAGAAGTCGGTGGACTTCAAGAGAGTCCACCGACTTCTTGGCTTTTTTTGGAAG-3′

RPC/EBPalpha::

5′-IndexTermTCGACTTCCAAAAAAAGCCAAGAAGTCGGTGGACTCTCTTGAAGTCCACCGACTTCTTGGCTGGG-3′

The non-complementary 9-nt loop sequences are underlined, and each sense oligonucleotide harbors a stretch of T as a PolIII transcription termination signal. The oligonucleotides were annealed and inserted 3′ of the H1-RNA promoter into the BglII/SalI-digested pBlueScript-derived pH1-plasmid to generate pH1-FMIP and pH1-CEBPalpha as described (Scherr et al., 2003). The control plasmid pH1-GL4 has been described earlier (Scherr et al., 2003). Finally, the H1-FMIP and H1-CEBPalpha expression cassette was excised by digestion with SmaI and HincII and blunt-end ligated either into the SnaBI site of the pdc-SEW lentiviral vector to generate pdcH1-FMIP-SEW or into the SnaBI site of the pdc-SR lentiviral vector to generate pdcH1-C/EBPalpha-SR plasmid. The lentiviral plasmid encodes RFPEXPRESS as reporter gene. Preparation of recombinant lentiviral supernatants and transduction were performed as described previously (Scherr et al., 2002). The titers were averaged and typically ranged between 1 and 5 × 108 IU/ml. Concentrated viral supernatants were used for transduction of 1 × 105 C2C12 cells in 48-well plates. After limiting dilution, positive colonies were selected by fluorescence microscopy (Scherr et al., 2002).

RNA isolation, Northern blot and real-time RT–PCR

Total RNA was isolated using RNeasy mini-spin columns (Qiagen, Hilden, Germany). Northern blot analysis was performed as described previously (Helftenbein et al., 1996). For RT–PCR, double-stranded cDNA was synthesized from 1 μg of total RNA using the cDNA Synthesis System (Roche Diagnostics, Penzberg, Germany) according to the manufacturer's instructions. PCR was performed using the primer sets described by Shi et al. (2003).

Immunoblotting

Western blotting was carried out as described previously (Mancini et al., 2004; Koch et al., 2005). Corresponding proteins were visualized by incubation with peroxidase-conjugated anti-goat, -mouse or -rabbit immunoglobulin followed by incubation with SuperSignal West Fento Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). Results were documented by LAS3000 (Fujifilm) and the intensity of bands was quantified using the program ‘Aida’. For quantification, we performed immunoblotting using 125I-labeled anti-rabbit IgG as a second antibody (Koch et al., 2005) to compare data obtained from the two methods.

Immunostaining

Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed in 3% formaldehyde and then treated with isopropanol. After washing with PBS, cells were incubated with C/EBPalpha-specific antibody, followed by incubation with FITC-conjugated anti-rabbit IgG.

Accession codes

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

We thank Regina Wilms, Sabine Klebba-Faerber and Iris Dallmann for technical assistance, Renate Scheibe for providing C2C12 cells and Bruce Boschek for critically reading the manuscript. The research was supported by Sonderforschungsbereich566 (B2) and the Deutsche Forschungsgemeinschaft (Ta-111/8/-4). AHB, KP, ADW and LL are supported by the Leukaemia Research Fund and ADW by BBSRC. This work is a part of PhD thesis of A-FN.

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Correspondence to T Tamura.

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Mancini, A., El Bounkari, O., Norrenbrock, A. et al. FMIP controls the adipocyte lineage commitment of C2C12 cells by downmodulation of C/EBPalpha. Oncogene 26, 1020–1027 (2007) doi:10.1038/sj.onc.1209853

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Keywords

  • adipocyte/muscle differentiation
  • insulin signaling
  • C/EBP
  • mesenchymal stem cells
  • mRNA export complex

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