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Previous Article | Next Article 
Molecular and Cellular Biology, July 2006, p. 5421-5435, Vol. 26, No. 14
0270-7306/06/$08.00+0 doi:10.1128/MCB.02437-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720
Received 21 December 2005/ Returned for modification 2 February 2006/ Accepted 20 April 2006
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INTRODUCTION |
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(TGF-), heparin-binding EGF-like growth factor (HB-EGF), and amphiregulin. All of these function through the EGF receptor, as well as Notch receptor and Notch ligands, such as Delta, to control cell fate during embryonic development. Pref-1 is highly expressed in preadipocytes, but its expression is extinguished during 3T3-L1 differentiation into adipocytes. Pref-1, therefore, is used as a unique marker for preadipocytes (30, 70). The importance of Pref-1 in vivo has been demonstrated through the generation of Pref-1 knockout mice and Pref-1 transgenic mice (44, 53). Pref-1 knockout mice display growth retardation, skeletal malformation, and accelerated adiposity. The absence of Pref-1 can also result in liver abnormalities and affect the development and function of B lymphocytes of hematopoietic origin (53). Conversely, mice overexpressing Pref-1 in adipose tissue show a decrease in adipose tissue mass, reduced expression of adipocyte markers, and lower adipocyte-secreted factors, including leptin and adiponectin. Due to decreased adipose tissue development, these mice suffer from hypertriglyceridemia, decreased glucose tolerance, and lower insulin sensitivity (44). Furthermore, Pref-1 has been shown to be an imprinted gene, paternally expressed via differential methylation of paternal and maternal alleles (63, 72, 77). In the adult stage, Pref-1 expression is restricted to preadipocytes and several neuroendocrine type of cells, but during the embryonic stages Pref-1 is found in multiple tissues (21, 35, 42, 55). Abnormalities detected in Pref-1 knockout and transgenic mice suggest that Pref-1 may be involved in the control of differentiation processes of several different cell types and have multiple functions during development. Pref-1 has recently been implicated in the maintenance of hepatoblasts, as well as osteoblasts (2, 28, 74).
Proteolytic cleavage of cell surface proteins, or ectodomain shedding, is an important mechanism whereby cells can regulate the repertoire of proteins expressed on their surface, e.g., shifting proteins from their membrane forms to soluble forms with modified biological function or availability. Several types of membrane proteins undergo ectodomain shedding. Those include growth factors, cytokines, cytokine receptors, and adhesion molecules (14, 25, 36, 48). EGF-like repeat-containing proteins including EGF, tumor necrosis factor alpha (TNF-), Notch, and Delta are processed by proteolysis (59). Processing of EGF receptor ligands such as EGF, TGF-, HB-EGF, and amphiregulin leads to availability of growth factors found in extracellular biological fluids. Similarly, we found that the membrane form of Pref-1 is proteolytically processed at two sites in the extracellular domain: one located near the fourth EGF repeat and the other in the region proximal to the transmembrane domain, resulting in the 50-kDa large and 25-kDa small soluble forms (65). We reported that only the 50-kDa large soluble form is active and sufficient for the inhibition of adipocyte differentiation (50). In this regard, of the four major alternate splicing products of Pref-1 (Pref-1A, -B, -C, and -D), Pref-1C and Pref-1D do not contain juxtamembrane cleavage domain due to the in-frame deletion and therefore do not produce the large soluble form.
Proteases, often called sheddases or secretases, are responsible for ectodomain shedding. Activation of intracellular second messenger systems, such as protein kinase C (PKC) or intracellular Ca2+ pathways, upregulates the activity of membrane sheddases. It is now known that shedding of EGF receptor ligands can be triggered by phorbol 12-myristate 13-acetate (PMA), by calcium ionophores, by serum, or by pervanadate (which inhibits tyrosine phosphatase activity) (4, 5, 19, 56). While PMA acts by PKC activation, serum and pervanadate act through PKC-independent pathway(s) (45). Cell surface proteolysis appears to be mediated by Ca/Zn-dependent endoproteases, matrix metalloproteinases (MMPs; also known as matrixins), and the closely related Zn-dependent "A disintegrin and metalloproteinases" (ADAMs) (12-14, 51, 57, 76). The MMPs are the primary matrix degrading proteases, and together they are able to degrade all protein components of the extracellular matrix, individual MMPs possessing distinct but overlapping substrate specificities. ADAMs are type I transmembrane proteins that contain a disintegrin-like, as well as metalloproteinase-like, domain. ADAMs play important roles in fertilization, epithelial and neural development, myoblast fusion, and angiogenesis. The first and the most well-characterized ADAM is TNF- converting enzyme (TACE; ADAM17), which has been implicated in the shedding of a number of diverse cell surface proteins, including TNF-, TNF receptors I and II, TGF-, L-selectin, and M-CSF receptor 1 (52). TACE knockout mice are not viable and TACE-deficient cells display impaired basal and stimulated the cleavage of many of these proteins (11, 18, 20, 31, 32, 57, 71). The tissue inhibitors of metalloproteinases (TIMPs) are considered to be the key endogenous inhibitors involved in regulation of the activity of MMPs and ADAMs, and some tissue inhibitors can associate with the cell surface (8, 43). Of the four distinct TIMPs (TIMP-1 to -4), TIMP-3 is known to be a good inhibitor of TACE (3, 10, 54).
Although only the 50-kDa soluble Pref-1 but not the membrane form of Pref-1 inhibits adipocyte differentiation, the molecular events leading ectodomain cleavage of the transmembrane form to the soluble form are currently not understood. In the present study, we examined the cellular processing of Pref-1 and the mechanisms that lead to the release of the soluble ectodomain. We show constitutive and inducible cleavage of Pref-1 from the cell surface by TACE. We also demonstrate the biological consequence of TACE-mediated Pref-1 cleavage on adipocyte differentiation.
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MATERIALS AND METHODS |
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21, which has deletion of 21 amino acids (283 to 303) in its juxtamembrane region that corresponds to the membrane proximal processing site (50). The open reading frames were subcloned into pLenti6/V5-D-TOPO vector (Invitrogen) by using PCR. Each of the viral expression constructs was tagged with HA in the ectodomain and with V5 at the C terminus. Plasmid containing the TACE cDNA sequence was kindly provided by C. Blobel (Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY). Myc-tagged TACE expression vector was constructed by adding Myc epitope (MEQKLISEEDL) at the C terminus of full-length TACE sequence and by subcloning it into pLenti6/V5-D-TOPO vector. The structures of pLenti-Pref-1A, pLenti-Mutant Pref-1A, and pLenti-TACE were confirmed by sequencing. To generate viral stocks, 293FT cells were cotransfected (Lipofectamine 2000) with the pLenti-Pref-1A, pLenti-Mutant Pref-1A, or pLenti-TACE vector (3 µg) and a mixture (9 µg) of the three packaging plasmids (pLP1, pLP2, and pLP/VSVG), which supply in trans the structural and replication proteins required for production of the lentivirus. At 72 h after transfection, the supernatants were harvested, filtered, and stored at –80°C.
Cell culture, lentivirus infection, and plasmid transfection. The shedding-defective CHO-M2 and its parental CHOT cells (4, 5) were kindly provided by J. Massague (Memorial Sloan-Kettering Cancer Center). Pref-1 knockout mouse embryo fibroblasts (MEFs) were isolated from E13.5 Pref-1 knockout embryos produced by mating homozygous Pref-1 knockout mice (1). Briefly, mouse embryos were dissected from the uterus and washed twice in fresh phosphate-buffered saline to remove any blood, followed by the removal of hearts and livers. The embryos were pooled and dissected into small pieces, suspended in 0.25% trypsin-EDTA (Invitrogen), and incubated for 30 min at 37°C. The tissues were triturated with a 10-ml pipette to dissociate. The resulting small clumps of cells were incubated in Dulbecco modified Eagle medium (DMEM). The MEFs were expanded twice before subjecting them to adipocyte differentiation.
All cells and cell lines were maintained in DMEM containing 4,500 mg of glucose/liter, 0.1 mM nonessential amino acids, 2 mM L-glutamine, and 10% fetal bovine serum (FBS; Omega) and incubated at 37°C with 5% CO2.
At 25% confluence, COS, CHO-M2, and CHOT cells were infected for 18 h with lentivirus containing HA-tagged Pref-1A plasmids (Lenti-Pref-1A, 1:500 dilution of viral supernatants in DMEM containing 10% FBS). The cells were then kept in selection media with blasticidin (5 µg/ml) for 10 days. The selection media were changed every 2 days. The pools of selected cells were used for detection of the expression of Pref-1A and shedding. The 3T3-L1 cells and MEFs at 70 to 80% confluence were infected with the lentivirus containing Pref-1A or TACE (Lenti-TACE, 1:100 dilution of viral supernatants) for 18 h, and the cells were maintained in DMEM with 10% FBS for further experiments. TACE target-specific small interfering RNA (siRNA; sc-36605) and the control siRNA (sc-36869; Santa Cruz) were transfected into 50% confluent COS cells or 70% confluent MEFs by using transfection reagent according to the manufacturer's instructions (Santa Cruz). At 30 h posttransfection, the cells were used to detect shedding and differentiation into adipocytes.
Analysis of shedding of extracellular domain of Pref-1A.
Confluent COS cells stably expressing Pref-1A were washed twice with serum-free DMEM and incubated in serum-free DMEM containing either no addition or one of the following agents: 100 µM H2O2, 100 µM pervanadate (V2O7), 1 µM calcium ionophore (A23187), 0.1% dimethyl sulfoxide (DMSO), or 1 µM PMA. V2O7 was freshly prepared (a 1:1 mixture of 100 mM vanadate [Fisher] and 100 mM H2O2) and used within 5 min. COS cells stably expressing Pref-1A were pretreated with 30 µM GM 6001 (Calbiochem) or DMSO (0.2% [vol/vol]) for 15 min at 37°C. Cells were then incubated in the presence or absence of 30 µM GM6001, 1 µM PMA, or DMSO for an additional 60 min. Confluent COS cells stably expressing Pref-1A were incubated in serum-free DMEM containing 100 µM TNF- proteinase inhibitor 0 (TAPI-0), 5 µg of TIMP-1/ml, 5 µg of TIMP-2/ml, or 5 µg of TIMP-3/ml (all from Calbiochem) with or without 1 µM PMA for 60 min. The supernatants and cell lysates were used for Western blot analysis.
Adipocyte differentiation assay. After transfection with siRNA or infection of lentivirus, 3T3-L1 cells and MEFs at 2 days postconfluence were used for the differentiation assay. Cells were treated with differentiation-inducing medium containing 1 µM dexamethasone, 0.5 mM methylisobutylxanthine, 1.67 µM insulin in DMEM with 10% FBS and 0.1% DMSO or 10 µM GM6001. The 3T3-L1 cells were incubated with differentiation media for 2 days. The cells were maintained in the DMEM containing 10% FBS without drugs for an additional 2 days for morphological observation, Oil red O staining, and RNA extraction. MEFs were kept in the differentiation media for 8 days, and the media were changed every 2 days. For Quantification, Oil red O stain was eluted from each well by adding 100% isopropanol. The optical density was measured at 500 nm and was expressed as the percentage of that from control 3T3-L1 cells.
Western blot analysis. After infection or transfection, cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% sodium dodecyl sulfate (SDS). Cell lysates and conditioned media were fractionated by SDS-10% polyacrylamide gel electrophoresis and transferred to Protran nitrocellulose membranes (Schleicher & Schuell) in transfer buffer containing 25 mM Tris-HCl, 192 mM glycine, and 20% methanol. After blocking with 4% nonfat dry milk in Tris-buffered saline-Tween 20 (TBST) buffer (10 mM Tris-HCl [pH 8.0], 165 mM NaCl, 0.05% Tween 20) for 1 h, the membranes were incubated overnight at 4°C with a 1:2,000 dilution of mouse anti-HA monoclonal antibody (Covance), a 1:1,000 dilution of rabbit anti-TACE polyclonal antibody (ProSci), and a 1:2,000 dilution of mouse anti-Myc monoclonal antibody (Upstate). The membranes were then washed twice with TBS for 15 min each time and then with TBST for 15 min, followed by reaction for 20 min with horseradish peroxidase-conjugated secondary antibody (Bio-Rad). V5 epitope was detected by using horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen). After washing, signals were detected by using a chemiluminescence system (Perkin-Elmer) and visualized by autoradiography.
Northern blot analysis and reverse transcription-PCR (RT-PCR).
Total RNA was extracted from cultured cells by using TRIzol reagent (Invitrogen). A 6-µg portion of total RNA was denatured with 6% formamide, subjected to electrophoresis on a 1.0% agarose-formaldehyde gel, and blotted onto Hybond-N membranes (Amersham). The membranes were hybridized with 32P-labeled cDNA to CCAAT/enhancer-binding protein (C/EBP) and peroxisome proliferator-activated receptor 
(PPAR). Hybridization was performed as described previously (53).
A total of 1 µg of total RNA from mouse tissues and cultured cells was used for RT. Using the cDNAs, PCRs were carried out at various cycles. The primer sets for adipocyte-specific secretory factor (ADSF)/resistin, adiponectin, fatty acid synthase (FAS), leptin, and ß-actin used for PCR were as described previously (50). The primer sets used for TACE were 5'-ATG AGG CGG CGT CTC CTC ATC CTG-3' and 5'-TCT CTT CAC TCG ACG AAC AAA CTC-3'.
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RESULTS |
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To examine the time course of PMA-stimulated Pref-1A shedding, cells stably expressing Pref-1A were treated with PMA for 5 to 120 min. The supernatants and the lysates from cells at various time points were used for Western blot analysis. In total cell lysates, the level of the full-length of Pref-1A detected was not significantly different in these samples, probably reflecting the fact that only a minor portion of Pref-1A undergoes processing. In the media, however, the 50-kDa soluble form of Pref-1A was detected at 30 min after the PMA treatment, in comparison with control samples from the cells treated with carrier DMSO (0.2% [vol/vol]) for 120 min. PMA treatment caused a rapid and marked increase in the release of the 50-kDa large soluble form, indicating that the release was time dependent (Fig. 2A). When we treated the cells with various concentrations of PMA from 0.01 to 10 µM, we detected a significant release of 50-kDa soluble Pref-1 at 1 µM PMA, and the release was further increased up to 10 µM PMA (Fig. 2B). Therefore, we used 1 µM PMA in all subsequent experiments. We also observed that the constitutive and PMA-induced Pref-1 ectodomain shedding were partially blocked by MAPK/ERK kinase (MEK) inhibitors PD98059 or U0126, an indication of the involvement of MAPK activation in the release of Pref-1 ectodomain (data not shown).
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21, by deleting the cleavage site proximal to the membrane so that the membrane form cannot be processed to generate the 50-kDa large soluble form. In the present study, we constructed a lentiviral expression vector for the same mutant Pref-1A but with an HA epitope tag inserted into the extracellular domain, as well as V5-tag in the C terminus (Fig. 1A). As expected, cells expressing wild-type Pref-1A showed both basal and PMA-stimulated release of the 50-kDa soluble Pref-1 in the media. In contrast, COS cells expressing the mutant Pref-1A showed no basal or PMA-stimulated release of soluble Pref-1A from the cell surface (Fig. 3C). These data indicate that the proximal cleavage site in the juxtamembrane domain is required for both the constitutive and the PMA-stimulated shedding of 50-kDa Pref-1 ectodomain.
TACE-mediated Pref-1 ectodomain shedding.
The marked sensitivity of PMA-induced Pref-1A shedding to inhibition by GM6001 suggested that MMPs or ADAM-family proteases are likely to be involved in Pref-1 ectodomain shedding. To characterize the protease activity involved in Pref-1 shedding, we examined the effects of the TIMPs, which are physiological protease inhibitors of MMPs and ADAMs. Most MMPs are inhibited by TIMP-1, TIMP-2, and TIMP-3, whereas ADAM-10 is inhibited by TIMP-1 and weakly by TIMP-3. On the other hand, TACE is well inhibited by TIMP-3 only (15, 64). COS cells stably expressing Pref-1A were incubated with TIMP-1, TIMP-2, or TIMP-3 in the presence of PMA. The release of Pref-1 ectodomain was inhibited only in the presence of TIMP-3 but not in the presence of TIMP-1 or TIMP-2 (Fig. 3D). This indicates that MMPs probably are not involved in Pref-1 cleavage and that TACE may regulate this process. We also used TAPI-0, which, although not specific for TACE, is extensively used to block TACE-mediated TNF- shedding. As shown in Fig. 3D, TAPI-0 treatment blocked the PMA-induced release of soluble Pref-1 in COS cells. Inhibition of the cleavage of Pref-1 ectodomain by TIMP-3, as well as by TAPI-0, strongly suggests that the protease for Pref-1 shedding could be TACE. In this regard, TACE is known to be involved in shedding not only of TNF- but also of various cell surface proteins, including EGF receptor ligands, that all contain EGF-repeats, such as TGF-, HB-EGF, and amphiregulin, but not EGF itself (16, 71).
To further determine the possible involvement of TACE in the shedding of the Pref-1A ectodomain, we infected COS cells stably expressing Pref-1A with the lentivirus packaged with Myc-tagged TACE expression vector (Fig. 4). Robust expression of TACE was detected by anti-Myc antibody in cells infected with the TACE expression virus. At 48 h postinfection, the control and TACE-expressing Pref-1 cells were subjected to PMA treatment. The constitutive and regulated Pref-1A shedding was significantly higher in cells infected by lentivirus containing TACE compared to the cells infected by the lentivirus containing control vector (Fig. 4A). This is evidence for TACE-mediated Pref-1A shedding in COS cells.
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Lack of basal and PMA-stimulated shedding of Pref-1 ectodomain in CHO-M2 cells with TACE mutation.
We next tested the shedding pattern of Pref-1A in CHO-M2 cells. CHO-M2 cells were originally isolated on the basis of the loss of TGF- shedding in response to PMA stimulation. The lack of ectodomain shedding of TGF- was recently reported to be due to mutations in the metalloprotease and cysteine-rich disintegrin domains of TACE (46). CHO-M2 cells, as well as parental CHOT cells, at 25% confluence were infected with Lenti-Pref-1A virus. After selection, drug-resistant cells were verified for the Pref-1A expression. Full-length Pref-1A was detected in the cell lysates by using anti-HA antibody (Fig. 5A). These CHO-M2 and CHOT cells stably expressing Pref-1A were used for the comparison of Pref-1A shedding. As observed above in COS and 3T3-L1 cells, Pref-1A in CHOT cells was constitutively cleaved efficiently by releasing the 50-kDa soluble form into the media. The release was stimulated by PMA treatment. In contrast, in the CHO-M2 mutant cells, although a similar level of membrane Pref-1A was detected compared to the CHOT cells, there was no detectable soluble Pref-1 in the media. Furthermore, the addition of PMA had no effect on Pref-1A cleavage (Fig. 5B). These data show that mutation in the TACE gene results in the loss of basal, as well as PMA-induced, Pref-1A shedding, demonstrating a requirement for TACE in Pref-1 ectodomain shedding.
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and C/EBP, as well as ADSF/resistin adiponectin and leptin as late markers of adipocyte differentiation. The mRNA levels for these adipocyte markers were highly induced in GM6001-treated cells and in the control lentivirus-infected cells. On the other hand, in TACE-transduced cells, the mRNA levels for adipocyte markers were markedly decreased (Fig. 7A, lower panel). These data suggest that increased TACE expression inhibits 3T3-L1 adipocyte differentiation and that this may be due to increased shedding of Pref-1 and generation of the 50-kDa soluble Pref-1 into the media.
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We next used 3T3-L1 cells to evaluate the effect of Pref-1 shedding by TACE on adipocyte differentiation. 3T3-L1 cells were infected with control lentivirus or Lenti-TACE virus, as well as control siRNA or TACE siRNA. These cells were then treated with differentiation agents. Compared to the control cells, which showed ca. 50% differentiation, cells infected with Lenti-TACE virus showed a lower degree of differentiation into adipocytes (20%), whereas cells transfected with TACE siRNA showed a higher degree of differentiation (70%). Similar differences in the degree of differentiation of these cells were detected by the Oil red O staining for lipid accumulation as shown in Fig. 7C, upper panel, as well as by the quantification of lipid accumulation (data not shown). Northern blot and RT-PCR analysis for adipocyte marker expression also demonstrated similar differences in the degree of differentiation. As shown in Fig. 7C, lower panel, we observed decreased expression levels of adipocyte markers, PPAR and C/EBP, ADSF/resistin, adiponectin, and FAS in cells infected with Lenti-TACE virus and increased levels of these markers in cells transfected with TACE siRNA. These data suggest that increased TACE expression enhances the inhibitory effect of Pref-1 on the adipocyte differentiation in 3T3-L1 cells, whereas knockdown of TACE expression blocks the inhibitory effect of Pref-1 on adipocyte differentiation.
Pref-1 knockout (KO) MEFs, which are deficient in Pref-1, provide an ideal model system to examine the effects of Pref-1 on adipocyte differentiation. As shown in Fig. 8A, left panel, Pref-1 mRNA was easily detected in wild-type MEFs but not in Pref-1 KO MEFs isolated from Pref-1 KO mice that we previously generated (53). TACE mRNA was found in both Pref-1 KO and wild-type MEFs at similar levels. We next compared Pref-1 KO MEFs and wild-type MEFs for the effect of TACE knockdown on adipocyte differentiation. After control siRNA or TACE siRNA transfection, MEFs were treated with differentiation-inducing agents to allow adipocyte differentiation. Control siRNA-transfected cells expressed TACE, whereas TACE siRNA-transfected cells had very low, but barely detectable levels of TACE. As predicted, the adipogenic transcription factors PPAR and C/EBP were expressed at higher levels in Pref-1 KO MEFs transfected with control siRNA compared to the wild-type MEFs. On the other hand, the expression levels for these adipogenic transcription factors in Pref-1 KO MEFs and wild-type MEFs that were both transfected with TACE siRNA were found to be high, similar to the level found in Pref-1 KO MEFs that were transfected with control siRNA (Fig. 8A, right panel). These results indicate that, in the absence of Pref-1, TACE could not exert detectable inhibitory effect on adipocyte differentiation, an effect that is prominent in the wild-type MEFs expressing Pref-1.
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Using Pref-1 KO MEFs, we next attempted to understand more clearly whether TACE siRNA can abrogate the Pref-1 effect on adipocyte differentiation. In the absence of Pref-1, 90% of the Pref-1 KO MEFs differentiated into adipocytes upon treatment with differentiation-inducing agents (Fig. 9Aa and e). On the other hand, ca. 50% of the Pref-1 KO MEFs infected with Lenti-Pref-1A virus differentiated into adipocytes (Fig. 9Ab and f). As predicted, cells coinfected with Lenti-Pref-1A and Lenti-TACE virus (Fig. 9Ac and g) showed an even lower degree of differentiation into adipocytes (35%) compared to cells infected with only Lenti-Pref-1A virus. In contrast, cells infected with Lenti Pref-1A virus along with TACE siRNA (Fig. 9Ad and h) showed higher degree of differentiation (80%) compared to cells infected with Lenti-Pref-1A virus only. Differences in the degree of differentiation of these cells were also shown in the staining with Oil red O (Fig. 9B, upper panel) or by quantification of the Oil red O staining (data not shown). Furthermore, the mRNA expression levels for adipocyte markers, PPAR, C/EBP, adiponectin, ADSF/resistin, FAS, and leptin were lower in the cells infected with Lenti-Pref-1A virus than in the cells transfected with control lentivirus or control siRNA. The mRNA levels for these markers were further reduced when the cells were infected with both Pref-1A and TACE lentivirus. In contrast, transfection of the TACE siRNA in Pref-1A-expressing cells increased the mRNA levels of these adipose markers and blocked the inhibitory effect of Pref-1 on adipocyte differentiation (Fig. 9B, lower panel). These data strongly indicate that the inhibitory effect of Pref-1 on adipocyte differentiation is enhanced by TACE overexpression but prevented by TACE siRNA.
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, HB-EGF, and amphiregulin precursors are regulated phenomena and are induced by PMA. Studies of HB-EGF and IL-6R suggest that PKC
is the target for phorbol ester-induced processing of HB-EGF and IL-6R (39, 75).
In the present study, we identified TACE as a protease responsible for Pref-1 shedding. Evidence for a role of TACE in regulating the shedding of Pref-1 came from the experiments in 3T3-L1 cells or in wild-type MEFs coexpressing Pref-1A and TACE. In these cells, endogenous Pref-1 is downregulated during adipocyte differentiation, whereas TACE is expressed throughout differentiation (Fig. 6). TACE-mediated Pref-1 shedding is demonstrated by the increased shedding of Pref-1A with the overexpression of TACE in both COS and 3T3-L1 cells and by the reduced shedding of Pref-1A with TACE inhibitors, including the physiological inhibitor of TACE, TIMP-3, as well as TAPI-0. However, the most definitive evidence for the role of TACE in Pref-1 cleavage came from the experiments with the mutant CHO-M2 cells, as well as TACE siRNA in testing the shedding of Pref-1A. CHO-M2 cells were isolated from ethyl methanesulfonate-treated CHO cells stably transfected with transmembrane TGF-. These cells express two TACE variants with distinct point mutations in the catalytic domain and cysteine-rich/disintegrin domain (46). In these CHO-M2 cells, we could not detect the basal or stimulated shedding of Pref-1A. The lack of shedding of Pref-1A in TACE-mutated CHO-M2 cells, as well as in the cells transfected with TACE siRNA, clearly demonstrates the involvement of TACE in the shedding of Pref-1A.
We also found that PMA-induced shedding of Pref-1A occurs by cleavage at the juxtamembrane region. The shedding of Pref-1A induced by PMA was efficiently abolished by the deletion of 21 amino acids in the juxtamembrane domain. These results indicate that this region is responsible for the shedding process by TACE. TACE activity has been known to be stimulated by phorbol esters (24), and the cytoplasmic tail of TACE can be phosphorylated at a specific site by extracellular signal-regulated kinase (ERK) (23). Although the regulation of TACE-mediated shedding is not clearly understood, the emerging theme is that stimulation of ADAM-mediated shedding by phorbol esters is mediated by PKC and the MEK/ERK pathway. However, phorbol ester stimulation can also be mediated independently of ERK via a yet-to-be-defined pathway (38). It has been known that treating preadipocytes with either phorbol esters or growth factors that can activate PKC inhibits adipocyte differentiation. While many isoforms of PKC are found in both preadipocytes and adipocytes, expression of the PKC, -, and - isoforms increases rapidly early during adipocyte differentiation, which subsequently decreases, resulting in lower levels in fully differentiated adipocytes (27, 49). By using antisense oligodeoxynucleotides to selectively deplete individual PKC subtypes, Fleming et al. reported that individual PKC isoforms may play distinct roles in the regulation of adipocyte differentiation. Among the PKC isoforms, PKC and - were shown to exert an inhibitory influence upon differentiation (27). The anti-adipogenic effect of PDGF was recently reported to be partially reversed by the inactivation of PKC (6). The expression pattern of Pref-1 during MEF differentiation into adipocytes is similar to that of PKC, -, and - isoforms (78). The Pref-1 shedding induced by PMA treatment leads us to predict that the inhibitory effect of PKC on adipocyte differentiation might be, at least partially, due to the activation of TACE that results in increased shedding of Pref-1.
The effect of MEK inhibitors on shedding of Pref-1A suggests that both constitutive and activated shedding of Pref-1 depend on signaling via the ERK/MAPK pathway but not p38 and JNK in COS cells (data not shown). This is in contrast to TGF- and TNF- shedding. There are reports that stimulated shedding of these two factors is ERK dependent, whereas the basal release has been shown to depend on p38 activity in CHO cells (26). Conflicting results have been reported on the function and importance of MAPK pathways in adipocyte differentiation. In a series of studies, it has been shown that ERK plays a positive role in adipogenesis. Using the oligonucleotide antisense strategy and an ERK inhibitor U0126, ERK has been shown to be required for 3T3-L1 adipocyte differentiation (62, 73). These studies also suggested the requirement of ERK for the expression of the adipogenic transcription factors, C/EBP, -ß, and -, as well as PPAR (7, 9, 58). To this end, MEFs and preadipocytes isolated from ERK1 knockout mice exhibited impaired adipogenesis (17). In contrast, others have reported the inhibition of adipocyte differentiation upon MAPK activation (22, 29, 37, 60). Phosphorylation of PPAR by MAPK was shown to cause a reduction of its transcriptional activity, which leads to the inhibition of adipogenesis (37, 60). Similar to findings reported for the ERK/MAPK pathway, contradictory observations—either a requirement for p38 or inhibition by p38 of adipocyte differentiation—have been reported. These contradictory effects of MAPK pathway may be explained by the model systems used that have differing requirement for adipocyte differentiation, such clonal expansion. In the present study, we show that Pref-1 shedding is partially dependent on the ERK activities in COS cells. Studies with preadipocytes that can undergo adipogenesis will further define the role of ERK in Pref-1 processing and its effect on adipocyte differentiation.
Adipocyte differentiation is a complex process, dependent upon the strict temporal regulation of multiple and interacting signaling events. The signaling ultimately leads to the modulation of the expression of an array of genes necessary for the attainment of the adipocyte phenotype. Many of the key hormonal regulators of this process have now been identified (33, 34, 47, 61, 69, 78). However, the roles of TACE or TACE substrates that are involved in adipocyte differentiation have not been previously studied. Here, we demonstrate the biological function of TACE in adipocyte differentiation through the function of TACE in Pref-1 shedding in the differentiation of 3T3-L1 cells and MEFs into adipocytes. As shown in Fig. 7, compared to control 3T3-L1 cells, cells overexpressing TACE could not undergo adipocyte differentiation, whereas GM6001, an inhibitor of metalloproteases, enhanced differentiation. We clearly show that after treatment with differentiation-inducing agents, Pref-1-null MEFs failed to differentiate into adipocytes when infected with Lenti-Pref-1A virus. Furthermore, the degree of differentiation was even less upon Lenti-TACE virus infection. On the other hand, TACE siRNA blocked the inhibitory effects of Pref-1. These data show that the inhibitory effect of Pref-1 on adipocyte differentiation is probably due to TACE-mediated shedding of Pref-1 ectodomain, generating the biologically active 50-kDa soluble form.
The fact that increased expression of TACE alone in Pref-1 KO MEFs causes a slight but detectable inhibition of adipocyte differentiation suggests that, in the absence of Pref-1, TACE may have substrates other than Pref-1 that can inhibit adipocyte differentiation upon cleavage. In this regard, TACE is known to mediate the cleavage of other precursor molecules, including TNF- and TGF-, and, by doing so, TACE may be a key protease of the cell surface pro-protein convertase machinery. TACE-deficient mice are not viable and show multiple developmental defects (57), suggesting that TACE plays an essential role in development. The fact that Pref-1 knockout mice and Pref-1-overexpressing transgenic mice also show multiple developmental abnormalities suggests that TACE-mediated cleavage of Pref-1 is likely to act as an important regulator of Pref-1 function and contribute to defects in TACE-null mice during development in vivo.
We conclude that Pref-1 might be a newly discovered TACE substrate. Pref-1 inhibition of adipocyte differentiation in vitro and the potential role of Pref-1 in development in vivo may be mediated by TACE-induced shedding. By TACE-mediated shedding, Pref-1 may function in an endocrine and/or paracrine manner in various developmental processes, including adipogenesis.
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ACKNOWLEDGMENTS |
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This study was supported by National Institutes of Health grant DK050828 (to H.S.S.).
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