Transforming Growth Factor-β Regulates Stearoyl Coenzyme A Desaturase Expression through a Smad Signaling Pathway

The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-β (TGF-β), a multifunctional cytokine, increasedSCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-β isoforms, β1, β2, and β3. However, SCD mRNA expression was not increased either by other members of the TGF-β family or by other growth factors or cytokines. TGF-β also increasedSCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-β-treated human retinal pigment epithelial cells. TGF-β did not induceSCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-β effect in this cell line. Moreover, TGF-β-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-β signal transduction pathway involving Smad proteins appears to regulate the cellular expression of theSCD gene, and this regulation may play an important role in lipid metabolism.

Stearoyl-CoA desaturase (SCD, 1 EC 1.14.99.5), a rate-limiting enzyme in the biosynthesis of unsaturated fatty acids, catalyzes the desaturation of stearic acid and palmitic acid into oleic acid and palmitoleic acid, respectively (1). The oxidative reaction catalyzed by this microsomal protein requires the participation of O 2, NADPH, cytochrome b 5 , and cytochrome b 5 reductase. Oleic and palmitoleic acids are the predominant unsaturated fatty acids present in fat depots and membrane phospholipids (2). The ratio of saturated to unsaturated fatty acids is thought to control the structural integrity and fluidity of membranes, which in turn modulate cell growth and differentiation (3,4). The regulation of SCD is of physiological importance because any changes in its activity can modulate the membrane fluidity and thereby alter cellular functions. Stearoyl-CoA desaturase has been shown to be regulated by dietary factors, hormones, metals, and peroxisomal proliferators (2,(5)(6)(7)(8)(9). In the mouse and rat, there are two Scd genes, Scd1 and Scd2 (10,11). Recently, a third Scd gene, Scd3, has been identified in mouse (12). Although the proteins encoded by these three genes show Ͼ87% sequence homology, they exhibit tissue-specific expression patterns, and transcriptional regulation, likely because of marked difference in their promoter sequences. However, in the human, SCD is expressed as a single gene that yields two transcripts, 3.9 and 5.2 kb in size, resulting from the alternative usage of polyadenylation sites present in the 3Ј-untranslated region (13). Both transcripts encode the same functionally active 359-amino acid protein with a molecular mass of 41.5 kDa. The human SCD promoter region has recently been characterized, and this gene is transcriptionally regulated by sterol regulatory binding protein, mono-and polyunsaturated fatty acids, and cholesterol (14,15). Recently, we have shown that the expression of SCD in human retinal pigment epithelial cells is regulated by retinoic acid (16).
The retinal pigment epithelium (RPE) is a polarized monolayer of highly differentiated epithelial cells, situated between the neurosensory retina and the vascularized choroid (17). It forms part of the blood-retinal barrier and is important for homeostasis of the outer retina. RPE cells provide nutrients to the photoreceptor cells and carry out phagocytosis and degradation of rod outer segment tips undergoing circadian shedding. These cells are also able to produce a variety of cytokines, which may play a role not only in the development, differentiation, and survival of retinal cells but also in several intraocular pathological conditions (18,19). Among these cytokines is transforming growth factor-␤ (TGF-␤), an anti-inflammatory growth factor that plays a pivotal role in ocular diseases such as proliferative vitreoretinopathy and age-related macular degeneration (20,21). It is thus of interest to know whether this cytokine regulates SCD expression.
The TGF-␤ family comprises a large group of multifunctional cytokines with widespread distribution. They have a broad range of biological effects including cell growth, differentiation, wound healing, apoptosis, immunomodulation, and stimulation of extracellular matrix formation (22,23). There are three mammalian TGF-␤-isoforms, TGF-␤1, TGF-␤2 and TGF-␤3, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
In this study we have demonstrated for the first time that SCD expression is induced by TGF-␤ and that this induction is mediated through a signal transduction pathway involving Smad proteins.
Cells and Culture Conditions-Cultures of primary human retinal pigment epithelial (HRPE) cells were established from explants derived from human donor eyes as described previously (33). The cells grown in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum, nonessential amino acids, penicillin (100 units/ml), streptomycin (100 g/ml), and amphotericin B (Fungizone, 250 ng/ml). Cells were grown to confluence in a complete medium, and the cultured cells were washed twice with serum-free medium and allowed to grow overnight in the same medium. The culture medium was replaced next day with fresh serum-free medium and was incubated in serum-free medium with or without TGF-␤ and other cytokines for the indicated time intervals. All experiments were performed with cells between passages 6 and 12. Human corneal epithelial cells (HCE-T), corneal and choroidal fibroblasts (HCRF, HCHF), and human lung fibroblasts (MRC-5) were grown in minimum essential medium containing 10% fetal bovine serum. Human breast cancer cell line (MDA-MB-468), and hepatocellular carcinoma cell line (HepG2) were also grown in the same medium as described above. The cells were maintained at 37°C in a humidified environment of 5% CO 2 .
Adenoviral Vector and Infection of Cells-Replication-deficient recombinant adenoviral vectors expressing FLAG-tagged full-length murine Smad4 and Smad7 under the control of a cytomegalovirus promoter were used (34). The optimal multiplicity of infection was determined using a lacZ control virus. Adenoviral stocks propagated in 293T cells were used to infect, MDA-MB-468 or HepG2 cells, respectively, for Smad4 and Smad7. Based on the ␤-galactosidase assay, both cells were infected with the corresponding adenoviral constructs at a multiplicity of infection of 100. The medium was changed after 3 h post-infection, and the cells were allowed to grow for 24 h. The cells were serum-starved overnight and treated with TGF-␤1 (5 ng/ml) for various time points, and SCD mRNA expression was analyzed.
RNA Extraction and Northern Blot Analysis-Total RNA was extracted from cultured cells using RNAzol B RNA isolation kit (Tel-Test, Friendswood, TX). Equal amounts of RNA samples were electrophoresed on a 1% agarose gel containing 2% formaldehyde. The RNA bands from the gel were transferred to a Nytran membrane by capillary blotting (Schleicher & Schuell). Equal loadings of the gel and equal transfer efficiency of RNA samples were verified by ethidium bromide staining of 28 and 18 S rRNAs on the gel and the membrane. The cDNA probe for SCD was generated by reverse transcription-PCR from human brain RNA as described previously (16). The identity of the amplification product was confirmed by sequencing, and its sequence was found to be identical to base pairs 198 -710 of the SCD cDNA sequence (GenBank accession no. AF097514). The cDNA probe was labeled with [␣-32 P]dCTP and hybridized to the blot using QuickHyb hybridization solution (Stratagene, La Jolla, CA). Hybridization and washing were done according to the manufacturer's protocol. The blot was exposed to Kodak X-Omat AR film. The relative expression of SCD was quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Normalization of the signal intensity was done using the intensity of ethidium bromide-stained 18 S rRNA band. Membranes were stripped and reprobed with glyceladehyde-3-phosphate dehydrogenase cDNA probe (CLONTECH) when necessary.
Assay of Oleic Acid Formation in HRPE Cells-Oleic acid formation in HRPE cells was estimated as described previously (16). Briefly, the cells were grown in 100-mm culture dishes treated with TGF-␤ (5 ng/ml) for 12 h. [1-14 C]Stearic acid in fatty acid-free bovine serum albumin was added at this time, and the cells were allowed to grow for an additional 6 h. The cells were washed twice with 5 ml of ice-cold phosphate-buffered saline, pH 7.4, and then harvested in 2.0 ml of phosphate-buffered saline by the use of a rubber policeman. Cells were homogenized in a Potter-Elvehjem glass homogenizer; lipids were extracted with chloroform/methanol (2:1) by vortexing and centrifugation at 3000 rpm for 15 min. The lipid phase at the bottom was collected, dried under argon, and analyzed for fatty acids using HPLC. The lipids were solubilized, saponified, and derivatized. The resulting samples were analyzed using a Hewlett-Packard HP1090 HPLC on a 4.6 ϫ 250-mm, 5.0-m C8 Supelco column in line with Packard A500 radiometric flow scintillation counter. The derivatized fatty acids were detected by absorbance at 250 nm. Individual fatty acids were identified by using labeled and unlabeled standards. Radioactive oleic acid was detected and quantified using Packard FLO-ONE software.
Western Immunoblot Analysis-The cultured human RPE cells with or without TGF-␤ treatment were washed twice with phosphate-buffered saline, and cell extracts were prepared as described previously (35). Protein concentrations of the cell extracts were quantified using Bio-Rad protein assay reagent. Equal amounts of total protein (50 g) from each sample were subjected to SDS-polyacrylamide gel electrophoresis using 10 -20% Tricine gels and then transferred to a nitrocellulose membrane (Invitrogen). The membranes were incubated for 1 h with anti-human Smad2 or anti-phospho-Smad2 (Ser 465/467) antibody. An anti-FLAG M2 monoclonal antibody (Sigma) was used to detect the adenoviral expression of Smad4 or Smad7 in MDA-MB-468 and HepG2 cells, respectively. Peroxidase-conjugated goat anti-rabbit IgG antibody (1:5000) was used as secondary antibody. Immunocomplexes were visualized by a chemiluminescence method using a Lumi-Light PLUS Western blotting kit (Roche Molecular Biochemicals).

TGF-␤1 Induces SCD mRNA Expression in HRPE Cells-We
studied the effect of TGF-␤ on SCD expression in HRPE cells in culture by Northern blot analysis. These cells have been shown previously to respond to TGF-␤ treatment (35,36). The expression of both SCD transcripts (3.9 and 5.2 kb) were greatly increased in HRPE cells treated with TGF-␤1 for 24 h (Fig. 1A). We also studied the effect of other growth factors and cytokines, such as interferon-␥, IL-1␤, IL-1␣, tumor necrosis factor-␣, TGF-␣, basic fibroblast growth factor, and platelet-derived growth factor. However, unlike TGF-␤1, none of these agents were effective inducers of SCD mRNA expression. The increased SCD mRNA expression induced by TGF-␤1 was effectively blocked by a neutralizing anti-TGF-␤1 antibody preparation (Fig. 1B). Thus, the observed effect was not because of any contaminant present in the TGF-␤1 preparation.
The effect of different TGF-␤ isoforms, ␤1, ␤2 and ␤3, on SCD mRNA expression was analyzed in HRPE cells ( Fig. 2A). Expression of both SCD transcripts was increased with all three TGF-␤ isoforms. The effect of other TGF-␤ family members on SCD mRNA expression was also studied (Fig. 2B). Unlike TGF-␤, BMP-4, activin A, and inhibin A were unable to induce SCD mRNA expression. This result clearly demonstrates the specificity of TGF-␤ on SCD mRNA expression in HRPE cells.
The effects of concentration and time on TGF-␤-induced SCD mRNA expression were analyzed (Fig. 3). The expression of both SCD transcripts increased with increasing concentrations of TGF-␤1. More than a 3-fold increase over the base line in SCD expression was observed at the optimal TGF-␤1 concentration of 5 ng/ml. The increase in SCD mRNA expression induced by TGF-␤1 was also time-dependent. Expression was increased within 2 h, and the maximum was observed at 18 h following treatment with TGF-␤1. SCD expression was not changed in untreated cells used as controls for the various time points (data not shown). It was of interest to see whether the TGF-␤1-induced increase in SCD mRNA expression in HRPE cells is translated into a corresponding increase in SCD enzyme activity. SCD enzyme activity was measured in HRPE cells in culture. Cells were incubated with [1-14 C]stearic acid for 6 h following a 12-h treatment with 5 ng/ml TGF-␤1. The lipids extracted from the cells were analyzed for oleic acid formation using HPLC. As shown in Fig. 4, the oleic acid formation in HRPE cells was increased to 216% after TGF-␤1 treatment. As expected, the treatment also resulted in an ϳ4-fold increase in SCD mRNA expression. This result shows that the increased SCD mRNA expression induced by TGF-␤1 in HRPE cells is associated with an increase in SCD enzyme activity.

TGF-␤1 Induces SCD mRNA Expression in Several Types of Cultured Human Cells-
The effect of TGF-␤1 on SCD mRNA expression was studied in various types of human cells in culture (Fig. 5). Apart from HRPE cells, the increased SCD mRNA expression induced by TGF-␤1 was also observed in ARPE-19, a well characterized human RPE cell line (37). We then tested cells derived from sources other than RPE. The TGF-␤1-induced elevation in SCD mRNA expression was also noted in corneal epithelial cells, corneal fibroblasts, choroidal fibroblasts, and lung fibroblasts. However, there was no appreciable induction of SCD mRNA expression in human amniotic epithelial cell line (WISH), where the basal expression level was already high. Thus, the regulation of SCD mRNA expression by TGF-␤1 is not limited to RPE cells but rather is present in multiple cell types.
Inhibition of TGF-␤1-induced SCD mRNA Expression in HRPE Cells by Actinomycin D-The effect of actinomycin D, an intercalating transcriptional inhibitor, on TGF-␤1-induced SCD mRNA expression was studied (Fig. 6). HRPE cells were treated with TGF-␤ in the presence of indicated concentrations of actinomycin D. Essentially complete inhibition of TGF-␤1induced increase in SCD mRNA expression is found even at 1 ng/ml, the lowest concentration of actinomycin D. Thus, the regulation of SCD mRNA expression by TGF-␤ appears to occur at the level of transcription rather than through stabilization of the message.
Increase in Smad2 Phosphorylation in HRPE Cells by TGF-␤-TGF-␤ signals from the cell surface to the nucleus are transduced by Smads 2, 3, and 4, and phosphorylation of Smad2 is often crucial for this downstream signaling cascade (27,28). Cell extracts from HRPE cells, treated with TGF-␤ (5 ng/ml) at various time points, were analyzed by Western blotting for phosphorylated Smad2 (upper panel) and total Smad2 (lower panel) using an anti-phospho-Smad2 antibody and an anti-Smad2 antibody, respectively (Fig. 7). The ϳ58-kDa Smad2 protein was present in both control and TGF-␤-treated HRPE cells, and an increase in phosphorylated Smad2 was noticed within 20 min of TGF-␤ treatment. This increase in Smad2 phosphorylation was transient and lasted for up to 60 min. Thus, Smad2 phosphorylation in HRPE cells is increased shortly after TGF-␤1 treatment, indicating that these cells can respond to the ligand through a Smad-dependent pathway.
Smad7 Overexpression Inhibits TGF-␤-induced SCD mRNA Expression-Overexpression of Smad7, an inhibitory Smad, has been shown to block TGF-␤-induced cellular responses (38,39). To demonstrate further that the increase in SCD expression occurs through a TGF-␤ receptor-mediated mechanism, overexpression of Smad7 was used to abrogate the induction of this message. As shown in Fig. 8A, both SCD transcripts were present in HepG2 cells, and their expression was up-regulated by TGF-␤ in a time-dependent manner. Smad7 overexpression was attained in these cells using an adenoviral construct. The overexpression of Smad7 was determined by Western blot using an anti-FLAG monoclonal antibody to the FLAG epitope

FIG. 4. Effect of TGF-␤1 on oleic acid formation in HRPE cells.
The cells were treated with or without 5 ng/ml TGF-␤1 for 12 h and incubated for an additional 6 h in the presence of 50 M [1-14 C]stearic acid. Total lipids were extracted, saponified, derivatized, and analyzed for oleic acid by HPLC. Panel A, the chart shows the oleic acid formation in control and TGF-␤1-treated cells. The dpm associated with oleic acid peak as well as the total dpm in the derivatized lipid factions were determined. The oleic acid formation is shown as percentage of total dpm. The values are the mean Ϯ S.D., n ϭ 3. *, p Ͻ 0.0001 treated versus control values. Panel B, total RNA preparations from control and TGF-␤1-treated cells were analyzed by Northern blotting using a SCD-specific cDNA probe. Ethidium bromide-stained 28 and 18 S rRNA bands as well as the blot reprobed with a cDNA probe specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown to indicate that similar amounts of RNA were loaded in different lanes. Panel C, the chart shows the relative intensities of bands on the Northern blot in panel B determined using a PhosphorImager. present in the construct (data not shown). TGF-␤-induced SCD mRNA expression was completely abolished by Smad7 overexpression (Fig. 8B). This result indicates that SCD expression induced by TGF-␤ is susceptible to inhibition by Smad7.
SCD mRNA Expression Induced by TGF-␤ Is Smad4-dependent-Because the expression of SCD is increased so substantially by TGF-␤, and because HRPE cells can activate a Smad pathway following stimulation by TGF-␤, we next sought to determine whether the increased expression of SCD by TGF-␤ requires the Smad pathway. Smad4, known as common partner Smad, is essential for the translocation of phosphorylated Smad2 and/or Smad3 to the nucleus, where these proteins regulate transcription of target genes (28 -30). The role of Smad4 in TGF-␤-induced SCD mRNA expression was analyzed using a human breast cancer cell line, MDA-MB-468, deficient in Smad4 (40). Two SCD transcripts (3.9 and 5.2 kb) were present in MDA-MB-468 cell line. However, no increase in SCD mRNA expression was observed in these cells as a result of TGF-␤ treatment (Fig. 9A). This indicates that a signal transduction pathway involving Smad4 is likely to be necessary for the TGF-␤1-induced SCD mRNA expression. If so, the overexpression of Smad4 should restore the TGF-␤1 effect on SCD mRNA expression in the Smad4-deficient cell line. SCD mRNA expression was analyzed in MDA-MB-468 after overexpression of Smad4 using an adenoviral construct. Expression of exogenous Smad4 was demonstrated in infected cells by Western blot analysis using anti-FLAG antibody (data not shown). SCD mRNA expression was increased in a time-dependent manner by TGF-␤ in Smad4 overexpressed MDA-MB-468 cells (Fig.  9B). Thus, Smad4 is involved in the induction of SCD mRNA expression by TGF-␤. DISCUSSION In the present study, we have shown for the first time that the expression of the SCD gene is regulated by TGF-␤. SCD mRNA expression in cultured human RPE cells is induced by all three TGF-␤ isoforms found in mammals. Other TGF-␤ family members, however, and several other unrelated growth factors and cytokines failed to elicit this response. We further determined that the regulation of SCD mRNA expression by TGF-␤ is not limited to RPE cells. Several cell culture systems tested responded to TGF-␤ treatment by increasing the expression of SCD mRNA. The increase in SCD mRNA expression in TGF-␤-treated HRPE cells was associated with an increase in SCD enzyme activity, as evidenced by the increased formation of oleic acid from stearic acid in treated cells. However, the increase in the SCD enzyme activity was much smaller than that expected from the increase in the SCD mRNA expression. It is interesting to note that a large increase in SCD mRNA expression observed in all-trans-retinoic acid-treated ARPE-19 cells, another human RPE cell culture system, was also not accompanied by a corresponding increase in SCD enzyme activity (16). This could be because of the translational deficiency associated with certain genes in cultured RPE cells. It has been reported that these cells under certain conditions express a large amount of RPE65 mRNA with no detectable amount of RPE65 protein (41).
The physiological role of the regulation of SCD expression by TGF-␤ remains to be elucidated. TGF-␤ is known as a potent regulator of many biological functions including cell growth, differentiation, and apoptosis (22,23). Interestingly, the regulation of SCD expression could also affect these cellular functions. An increase in the activity of SCD, an important regulatory enzyme in the synthesis of unsaturated fatty acids, could lead to increased formation of unsaturated fatty acids, oleic acid, and palmitoleic acid (1). This in turn modulates the ratio of saturated to unsaturated fatty acids in the cell membrane. This ratio is a key regulator of cell membrane fluidity and structural integrity (3,42). Alteration in the unsaturated fatty acid content in the membrane caused by changes in the SCD enzyme activity has been shown to control membrane fluidity (43). Cell membrane fluidity is thought to play an important role in cell growth, differentiation, and apoptosis (4,44). Change in Scd1 expression is associated with the differentiation of mouse preadipocytes to adipocytes (45). Increased oleic acid content is thought to be responsible for the accelerated cell growth, metabolism, and cell division associated with cancer cells (46). Thus, it is possible that the effect of TGF-␤ on cell growth, differentiation, and apoptosis could be partly mediated through the regulation of SCD expression.
It appears that the regulation of SCD gene expression by TGF-␤ is mediated through a Smad signaling pathway. Increased phosphorylation of Smad2 preceded the induction of SCD mRNA expression in TGF-␤-treated human RPE cells. Normally, Smad2 is predominantly localized in the cytoplasm of the unstimulated cells (47). TGF-␤ induces the phosphorylation of Smad2, which then forms a complex with Smad4 and subsequently translocates to the nucleus to regulate gene transcription (27,28). Transcriptional regulation of several genes such as those for heme oxygenase-1, plasminogen activator inhibitor-1, JunB, human type VII collagen, platelet-derived growth factor-B chain, and human ␣2 (I) procollagen by TGF-␤ has been shown to be mediated through a signal transduction pathway involving Smad proteins (38, 48 -52).
SCD mRNA expression in human breast cancer cell line MDA-MB-468 was not induced by TGF-␤ treatment. The TGF-␤ signal transduction pathway involving Smad proteins is not functional in this Smad4-null cell line (40). It has been reported that the overexpression of Smad4 can restore the TGF-␤ signaling in this cell line (40,53). The MDA-MB-468 cells overexpressing Smad4 responded to TGF-␤ treatment by increasing SCD mRNA expression. Several studies have demonstrated that Smad4 is necessary for the TGF-␤ signaling (30,54). Thus, Smad4 appears to mediate the induction of SCD gene expression by TGF-␤.
SCD expression induced by TGF-␤1 in human hepatocellular carcinoma cell line, HepG2, was completely suppressed by the overexpression of Smad7. Smad7, an inhibitory Smad, has been shown to attenuate TGF-␤ signal transduction through an in- tracellular negative feedback loop (32,38,39). Smad7 is thought to preferentially inhibit TGF-␤ signaling by preventing the phosphorylation of Smad2 and/or Smad3 by type I receptor (32,55). Thus, it appears that overexpression of Smad7 suppresses TGF-␤-induced SCD mRNA expression by interfering with the TGF-␤ signal transduction pathway involving Smad proteins.
Apart from the TGF-␤-specific Smad pathway, a parallel pathway involving mitogen-activated protein kinase (MAPK) has been reported for the regulation of certain genes by TGF-␤ (56). However, it appears that an MAPK pathway may not mediate the regulation of SCD gene expression by TGF-␤. PD98059 and SB203580 are known inhibitors of different phosphorylation cascades in the MAPK pathway (57). Both these compounds failed to inhibit the TGF-␤-induced SCD mRNA expression (data not shown).
The regulation of SCD mRNA expression by TGF-␤ appears to be at the level of transcription. Actinomycin D, a known transcriptional inhibitor, effectively blocked the TGF-␤-induced SCD mRNA expression. The transcriptional regulation by TGF-␤ is mediated through the binding of Smad proteins to the Smad-binding elements (SBE) present in the promoter regions of target genes (48 -52). The consensus sequence for SBE is the 8-base pair palindrome, GTCTAGAC (58,59). We have analyzed the promoter sequence of human SCD reported by Zhang et al. (14) and Bene et al. (15) for the presence of putative SBE elements. Although the canonical SBE sequence was not detected in the SCD 5Ј-flanking region, a 5-base pair CAGAC sequence was found in four different positions in the Ϫ1510 to Ϫ3056 base pair region. This 5-base pair sequence, also called Smad-binding element, is often present in the proximal region of genes regulated through Smad pathway (48,49,58). The 5Ј-flanking region of SCD also contains binding regions for SP1, AP-1 family members, and TFE3 at Ϫ410, Ϫ1665, and Ϫ3391 base pairs, respectively. These transcription factors are reported to cooperate with Smad proteins during the TGF-␤-induced transcriptional activation of target genes (60 -62). Further investigation using promoter-reporter constructs is necessary to identify which of these putative SBE is involved in the transcriptional regulation of SCD by TGF-␤.
The regulation of SCD expression by TGF-␤ may play an important role in the pathophysiology of the retinal pigment epithelium. TGF-␤ production in the eye is greatly increased during the retinal disorder known as proliferative vitreoretinopathy (20). Such an increase is also indicated in age-related macular degeneration (21). We have shown previously that retinoic acid increased the expression of SCD in human RPE cells (16). The relationship between the regulation of SCD by TGF-␤ and that by retinoic acid remains to be elucidated. Both TGF-␤ and retinoid signal transduction pathways are interrelated (63). Retinoic acid is reported to increase the expression of TGF-␤ and its receptors (64). The induction of a MUC4 gene expression by retinoic acid has been shown to be mediated through TGF-␤ (65). However, the effect of retinoic acid and TGF-␤ on the expression of CRBP-1 and ␣-smooth muscle actin in fibroblasts is independent or reciprocal, respectively (66). Both retinoic acid and TGF-␤ are known to regulate cell growth, differentiation, and apoptosis (23,67). Interestingly, SCD by its ability to control the cell membrane fluidity can also regulate these processes. Thus, our results imply that SCD could act as a common factor in mediating the actions of both retinoid and TGF-␤.
In summary, we have identified SCD as a gene regulated by TGF-␤ in human RPE cells and in several other cell lines. A signal transduction pathway involving Smad proteins appears to mediate the induction of SCD mRNA expression by TGF-␤. The regulation of SCD, a key regulatory enzyme in the lipid metabolic pathway, by TGF-␤, a pleiotropic cytokine, may play an important role in cellular pathophysiology and metabolism. Total RNA prepared at indicated time periods was analyzed by Northern blotting using a SCD cDNA-specific probe. Ethidium bromide-stained 28 and 18 S rRNA bands are shown to indicate that similar amounts of RNA were loaded in different lanes. Panel B, SCD mRNA expression in this Smad4-deficient cell line was induced by TGF-␤1 following Smad4 overexpression. MDA-MB-468 cells were transiently infected with an adenoviral construct expressing Smad4 as described under "Experimental Procedures." Total RNA preparations from cells treated with 5 ng/ml TGF-␤1 for indicated time intervals were analyzed by Northern blot for SCD mRNA expression.