Correspondence to: Rik Derynck, Department of Growth and Development, University of California at San Francisco, San Francisco, CA 94143-0640. Tel:(415) 476-7322 Fax:(415) 476-1499 E-mail:derynck{at}itsa.ucsf.edu.
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Abstract |
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TGF-ß inhibits adipocyte differentiation, yet is expressed by adipocytes. The function of TGF-ß in adipogenesis, and its mechanism of action, is unknown. To address the role of TGF-ß signaling in adipocyte differentiation, we characterized the expression of the TGF-ß receptors, and the Smads which transmit or inhibit TGF-ß signals, during adipogenesis in 3T3-F442A cells. We found that the cell-surface availability of TGF-ß receptors strongly decreased as adipogenesis proceeds. Whereas mRNA levels for Smads 2, 3, and 4 were unchanged during differentiation, mRNA levels for Smads 6 and 7, which are known to inhibit TGF-ß responses, decreased severely. Dominant negative interference with TGF-ß receptor signaling, by stably expressing a truncated type II TGF-ß receptor, enhanced differentiation and decreased growth. Stable overexpression of Smad2 or Smad3 inhibited differentiation and dominant negative inhibition of Smad3 function, but not Smad2 function, enhanced adipogenesis. Increased Smad6 and Smad7 levels blocked differentiation and enhanced TGF-ßinduced responses. The inhibitory effect of Smad7 on adipocyte differentiation and its cooperation with TGF-ß was associated with the C-domain of Smad7. Our results indicate that endogenous TGF-ß signaling regulates the rate of adipogenesis, and that Smad2 and Smad3 have distinct functions in this endogenous control of differentiation. Smad6 and Smad7 act as negative regulators of adipogenesis and, even though known to inhibit TGF-ß responses, enhance the effects of TGF-ß on these cells.
Key Words:
inhibitory Smads, PPAR, C/EBP, 3T3-F442A, retroviral vectors
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Introduction |
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TGF-ß regulates the differentiation program of a variety of cell types (for review see
While the effects of TGF-ß signaling on muscle, bone, and cartilage cell differentiation are well studied, little is known about how TGF-ß regulates adipocyte differentiation. Furthermore, the mechanism by which TGF-ß stimulates proliferation of mesenchymal cells ( (
Collectively, these observations suggest a role for endogenous TGF-ß in the development and function of adipose tissue. However, very little is known about the signaling mechanisms that lead to the differentiation responses to TGF-ß. TGF-ß family factors bind to a heteromeric cell-surface complex of two type II and two type I receptors (for reviews see
The expression and function of these components of the TGF-ß signaling pathway during adipocyte differentiation, and the role of endogenous TGF-ß expression and TGF-ß responsiveness in the regulation of adipocyte differentiation, are unknown. It is known that the block in differentiation induced by TGF-ß is accompanied by decreased mRNA levels for C/EBP and PPAR
(
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Materials and Methods |
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Construction of Expression Plasmids
Expression plasmids for the cytoplasmically truncated, dominant negative version of the type II TGF-ß receptor (dnTßRII;1 SSMS mutant was generated by removing the COOH-terminal sequence of Smad2 at the BamHI site, which is located 33 bp upstream of the stop codon, and replacing it with annealed oligonucleotides encoding the identical DNA sequence but lacking the last four codons. The cDNA for Smad3
SSVS was created by PCR amplification of the human Smad3 sequence without the last four codons, and was obtained from Y. Zhang (University of California, San Francisco, San Francisco, CA). The cDNAs for mouse Smad6 and mouse Smad7 were obtained from K. Miyazono (The Cancer Institute of Japanese Foundation for Cancer Research, Tokyo, Japan) and P. ten Dijke (Ludwig Institute for Cancer Research, Uppsala, Sweden), respectively. Mutant versions of mouse Smad7 containing a 19amino acid deletion at the COOH terminus (Smad7
C) or consisting only of the C-domain, amino acids 204426 (Smad7C), were obtained from P. ten Dijke.
Retroviral vectors were used to establish stable cell populations overexpressing components of the TGF-ß signaling pathway. The coding sequence for the dominant negative type II TGF-ß receptor, along with its COOH-terminal Flag tag, was subcloned into the HpaI site of the LNCX vector, which allows selection in the neomycin derivative G418 (SSMS, Smad3, and Smad3
SSVS were inserted into the HpaI site of LPCX. The coding regions for the NH2 terminally Flag-tagged Smad6, Smad7
C, and Smad7C were cloned into the BamHI-XhoI sites of pBabepuro3, whereas the coding region of Smad7 were cloned into the BamHI/blunt-ended EcoRI site of pBabepuro3.
Cell Culture and Generation of Stable Cell Lines
The preadipocyte cell line 3T3-F442A (
To generate retroviruses, Phoenix E cells were plated at 2.7 x 106 cells/60-mm tissue culture dish 24 h before transfection. Transfection was done using the calcium phosphate method (
Assay of Cell Growth Rates
Cells were trypsinized, resuspended in growth medium without selective antibiotic, and 2 x 104 cells were plated per well of 24-well dishes. The cells were washed twice with PBS on the following day, and then overlaid with DME containing 0.5% BSA (Sigma Chemical Co.) either without or with the indicated concentrations of TGF-ß. The next day, [3H]thymidine (2 Ci/mmol; NEN) was added to the medium at a concentration of 4 µCi/ml. Uptake of the label proceeded for 45 h. Cells were washed twice with PBS, fixed for 20 min with 10% TCA, washed twice with water, and solubilized for 20 min in 1 N NaOH. An equal volume of 1 N HCl was added, and the resulting lysate was subjected to liquid scintillation counting.
Analysis of Lipid Accumulation, RNA, and Protein
Neutral lipid accumulation was visualized by washing cell monolayers once with PBS, fixing for 15 min with buffered formalin, and staining them for 1 h in a freshly made solution containing four parts water mixed with six parts 0.5% Oil Red O (Sigma Chemical Co.) in isopropanol. Excess stain was removed, and the cells were washed several times with water.
RNA was isolated using the SV RNA isolation kit (Promega). 10 µg total RNA was denatured and electrophoresed in 1% formaldehyde gels, blotted to Biotrans nylon membranes (ICN), and hybridized to 32P-labeled cDNA probes, as described previously (-dCTP (6,000 Ci/mmol; NEN) by the random priming method (
, PPAR
2, ADD-1/SREBP1, aP2 and adipsin were obtained from B. Spiegelman (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). The cDNAs for C/EBPs ß and
were provided by S. McKnight (University of Texas, Southwestern Medical Center, Dallas, TX).
125I-labeled TGF-ß1 was purchased from NEN. Cross-linking to [125I]TGF-ß was performed as described (
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Results |
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Cell-Surface TGF-ß Receptors Decrease during Adipogenesis
3T3-F442A cells spontaneously undergo adipose differentiation, after reaching confluence in culture in the presence of FBS and insulin (
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The expression of TßRII and TßRI at the cell surface was assessed by cross-linking 125I-labeled TGF-ß to the cells (Fig 1 B). While preadipocytes expressed type III TGF-ß receptor (ßglycan), TßRII, and TßRI at the cell surface, the availability of these receptors strongly decreased during differentiation. These decreased levels were not a consequence of receptor occupation by unlabeled TGF-ß preventing [125I]TGF-ß binding since removal of endogenous ligand by suramin or acid washes did not enhance receptor binding of [125I]TGF-ß (data not shown). The total expression levels of the receptors could not be determined because of the low expression levels and limited quality of the available antibodies (data not shown). The strong decrease in cell-surface availability of the receptors during adipose differentiation, in contrast to the mRNA levels for TßRII and TßRI, indicates that the cells posttranscriptionally downregulate TGF-ß binding as they differentiate.
Regulation of Smad Expression during Adipocyte Differentiation
Smads act as intracellular effectors of TGF-ß signaling. Smad2 and Smad3 are activators of TGF-ß responses, and form heteromeric complexes with the common mediator Smad4. In contrast, Smad6 and Smad7 block TGF-ß responses. We assessed the mRNA expression of these Smads at different stages of adipose differentiation, both in TGF-ßtreated and untreated cells (Fig 2). In these experiments, the TGF-ßtreated cells failed to differentiate, as judged from the absence of lipid accumulation, although analysis of PPAR mRNA expression indicated that differentiation was not entirely blocked, but strongly decreased and delayed. Thus, PPAR
expression increased with time in TGF-ßtreated cells, but never achieved the expression level seen in untreated cells (Fig 2). All five Smads were expressed at all stages of differentiation of these cells. The Smad2 or Smad4 mRNA levels were unaffected by the differentiation stage or by TGF-ß, whereas Smad3 mRNA levels did not significantly change during differentiation, but were decreased by TGF-ß. This TGF-ßinduced repression decreased with the progression of adipose differentiation. Both Smad6 and Smad7 mRNAs decreased during differentiation, with Smad7 mRNA levels decreasing more abruptly than Smad6. In addition, TGF-ß increased Smad7 expression, but did not have a significant effect on Smad6 mRNA levels. We were unable to clearly detect endogenous Smad proteins, most likely because of poor antibody quality and/or low protein abundance (data not shown). Collectively, our data show that all TGF-ß Smads are expressed in 3T3-F442A cells, and that expression of the inhibitory Smads decreases during adipogenesis.
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Decreased TGF-ß Receptor Signaling Results in Accelerated Adipogenesis
Since the levels of cell-surface TGF-ß receptors and inhibitory Smads decreased during adipocyte differentiation, we determined the role of endogenous TGF-ß signaling and Smads in the growth and differentiation of these cells. We created cell lines, via retroviral transduction, which stably expressed individual components of the TGF-ß signaling system. First, we attempted to generate cells that would stably overexpress wild-type TßRII, to counteract the differentiation-linked suppression of cell-surface TGF-ß receptors. However, the virally expressed TßRII levels, while not decreased during differentiation as the endogenous receptors (data not shown), were too low to significantly change the overall receptor level or profile during differentiation. In contrast, we successfully generated cells that expressed a cytoplasmically truncated TßRII (dnTßRII), which acts as a dominant negative inhibitor of TGF-ß signaling (
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Using these cells, we evaluated the effect of dnTßRII expression on adipose differentiation, and in parallel, tested the effect of TGF-ß at 1 ng/ml, added 2 d before confluence and continuously present thereafter. At this dose, TGF-ß confers incomplete inhibition of adipose conversion (, ADD-1, PPAR
, and C/EBP
, as well as the later markers aP2 and adipsin. This study indicated that, while the extent of differentiation of dnTßRII and control cells was similar at day 8 after confluence, the rate of differentiation of dnTßRII cells was enhanced when compared with the parental cells (Fig 4 B). Thus, at day 3 after confluence, the mRNA levels for PPAR
, C/EBP
, aP2, and adipsin were significantly higher in dnTßRII cells than in control cells. In contrast, the mRNA levels of C/EBPß, C/EBP
, and ADD-1, earlier markers of adipogenic differentiation, were similar in both cell lines. The increased mRNA levels for PPAR
, C/EBP
, aP2, and adipsin in dnTßRII cells are consistent with the ability of TGF-ß to repress expression of these markers (
at similar levels as untreated control cells. Consistent with their impaired TGF-ß responsiveness, TGF-ßtreated dnTßRII cells showed only slight inhibition of the later differentiation markers.
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TGF-ß stimulates the proliferation of many types of mesenchymal cells, including 3T3-F442A preadipocytes (
Smad2 and Smad3 Differentially Regulate Adipogenesis and Cell Proliferation
To characterize the roles of Smad2 and Smad3 in adipocyte differentiation of 3T3-F442A cells, we generated two pairs of cell lines. One pair overexpressed wild-type Smad2 or Smad3, while the complementary pair expressed mutant versions of Smad2 (Smad2SSMS) or Smad3 (Smad3
SSVS). These mutants lacked the last four amino acids, including the COOH-terminal serines, and, thus, cannot be phosphorylated by the activated TßRI. Similarly to the 3SA derivatives of Smad2 and Smad3, in which the last three serines are mutated to alanine (
SSMS or Smad3
SSVS resulted in dominant negative interference of signaling by Smad2 or Smad3 in transfection/reporter assays (data not shown). Expression of wild-type and mutant Smad2 or Smad3 proteins could be readily detected in the stable cell populations (Fig 5 A). While we were unable to clearly detect endogenous Smad proteins, the viral transcripts were expressed at a >100-fold higher level than the endogenous Smad2 and Smad3 mRNAs (data not shown).
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We tested the abilities of these stable cell lines to undergo adipocyte differentiation and the ability of TGF-ß to inhibit their differentiation. Expression of either Smad2 or Smad2SSMS inhibited lipid accumulation, although the effect of Smad2
SSMS was weaker than Smad2 (Fig 5 B). Enhanced Smad3 expression resulted in a stronger inhibitory effect than Smad2 overexpression, while cells expressing Smad3
SSVS differentiated similarly to vector control cells (Fig 5 B). In the presence of 1 ng/ml TGF-ß, Smad2 or Smad3 expression both significantly augmented the differentiation-inhibiting effect of TGF-ß (Fig 5 C). However, the effect of Smad3 was stronger than Smad2; no lipid-accumulating cells were found in the Smad3-expressing cultures, whereas a few could be seen among the cells overexpressing Smad2. Cells expressing Smad3
SSVS differentiated to a much greater extent than control cells in the presence of TGF-ß, demonstrating that this Smad3 mutant blocked the differentiation-inhibitory effect of TGF-ß. In contrast, expression of Smad2
SSMS did not block this effect of TGF-ß, and slightly enhanced the inhibitory effect of TGF-ß (Fig 5 C).
The effect of these Smads on lipid accumulation was reflected in the expression pattern of adipocyte differentiation mRNAs (Fig 6 A). The induction of PPAR, ADD-1, C/EBP
, aP2, and adipsin was strongly reduced in cells with increased Smad3 expression, whereas the differentiation-dependent suppression of C/EBP
was abrogated. Smad2 or Smad2
SSMS expression resulted in only a slight decrease in the differentiation-dependent induction of the differentiation markers, whereas Smad3
SSVS expression did not significantly alter their expression. In the presence of TGF-ß, the Smad3
SSVS cells expressed PPAR
, C/EBP
, aP2, and adipsin at higher levels than in TGF-ßtreated control cells, suggesting that impaired Smad3 signaling suppresses the responsiveness to TGF-ß. In contrast, Smad2
SSMS expression enhanced the TGF-ßmediated suppression of these differentiation markers, although to a lesser extent than in cells overexpressing Smad2 or Smad3.
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We also measured the effect of overexpression of Smad2 or Smad3 or their dominant negative mutants on the growth rate (Fig 6 B). Expression of Smad2 or Smad2 SSMS had little if any effect on cell proliferation or its stimulation by TGF-ß. In contrast, Smad3 overexpression stimulated cell growth about threefold, both in the presence or absence of TGF-ß. Expression of Smad3
SSVS inhibited the growth rate in the presence of TGF-ß, and was ~30% less than the vector alone (Fig 6 B).
Finally, we evaluated the effect of TGF-ß on the morphology of these cell lines (Fig 7). Adipogenic conversion of control cells in the absence of TGF-ß resulted in rounding, accumulation of lipid droplets, and a consequent enlargement of the cells, whereas nearby cells that had not yet undergone differentiation maintained a fibroblastic appearance (Fig 7 A). TGF-ß treatment prevented cell rounding and lipid accumulation and conferred a more densely packed, spindly morphology to the cells (Fig 7 B). Increased expression of Smad2 (Fig 7 C) or Smad3 (Fig 7 D) enhanced these typical TGF-ßinduced morphological changes; however, the effect of Smad3 was stronger than that of Smad2, which is consistent with the milder effects of Smad2 versus Smad3 on TGF-ßinduced inhibition of lipid accumulation (Fig 5 C). Expression of Smad2SSMS conferred a morphology intermediate between the vector control and Smad2 overexpressing cells (Fig 7 E). In contrast, Smad3
SSVS expression counteracted the cell shape changes induced by TGF-ß and allowed a level of lipid accumulation (Fig 7 F), which approached that of control cells in the absence of TGF-ß (Fig 7 A). In the absence of TGF-ß, the morphology of the different Smad-expressing cell lines was similar to each other, except that the Smad3-expressing cells were somewhat more spindly and densely packed (data not shown), which is consistent with a constitutive level of TGF-ß signaling. These results identify Smad3 as a major regulator of adipogenic differentiation and of TGF-ßmediated inhibition of adipogenic conversion and growth stimulation. The Smad2 effects appear to be more complex and may reflect its involvement in only some TGF-ß effects, yet suggest that Smad2 is also an important regulator of normal adipocyte differentiation.
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Overexpression of Smad6 or Smad7 Blocks Adipogenesis and Enhances TGF-ßinduced Growth and Inhibition of Differentiation
In contrast to the roles of Smad2 and Smad3 as TGF-ß signaling effectors, Smad6 and Smad7 inhibit signaling by TGF-ß family members. We hypothesized that the overexpression of these proteins might result in a more complete blockade of TGF-ß signaling than expression of dominant negative Smad2 or 3 mutants expressed alone, or of dnTßRII. Furthermore, the downregulation of both Smad6 and Smad7 during adipocyte differentiation (Fig 2) suggested a function for these Smads in the differentiation process. Therefore, we evaluated the consequences of stable overexpression of these two inhibitory Smads. Retroviral expression of Smad6 exceeded by far the expression level of Smad7 (Fig 8 A). Expression of the viral mRNAs for these Smads was much higher than the corresponding endogenous Smad transcripts (data not shown).
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These cells were cultured under differentiation conditions, in the presence or absence of TGF-ß, to assess the effects of Smad6 or Smad7 on adipogenic conversion, and on the ability of TGF-ß to block differentiation. Expression of either Smad protein resulted in a profound blockage of lipid accumulation (Fig 8 B). Despite its lower expression level (Fig 8 A), the differentiation blockage induced by Smad7 was greater than that induced by Smad6. In the presence of TGF-ß, this inhibition was even more complete, and no adipocytes could be found in Smad7 overexpressing cell cultures treated with TGF-ß. These effects of Smad6 and Smad7 on adipose conversion were also apparent in the analysis of adipocyte marker expression (Fig 9 A). While C/EBPß levels were not strongly affected, Smad6 or Smad7 overexpression increased the levels of C/EBP, which is normally downregulated during differentiation. These cells also showed strongly reduced PPAR
, C/EBP
, aP2, and adipsin mRNAs, and a moderately reduced ADD-1 mRNA level. In the presence of TGF-ß, all the TGF-ßinduced changes in mRNA expression were enhanced by overexpression of Smad6 or Smad7.
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We next tested the effect of Smad6 or Smad7 overexpression on cell proliferation in the absence or presence of TGF-ß. Smad6 or Smad7 overexpression both greatly potentiated TGF-ßinduced growth stimulation (Fig 9 B) to a similar magnitude as Smad3 overexpression (Fig 6 B). However, in contrast to the effect of Smad3 overexpression, the basal growth rate in the absence of added TGF-ß was unchanged. Therefore, we conclude that Smad6 or Smad7 expression cooperates with TGF-ß to enhance cell proliferation, but not in the same manner as Smad3.
The Smad6- or Smad7-expressing cells had an unusual morphology. When subconfluent, the cells were overall similar in size, but the Smad6 and Smad7 cells had a more cuboidal appearance and smoother edges, and seemed more contact-inhibited than control cells (Fig 10 A). After reaching confluence under conditions that normally result in adipocyte differentiation, Smad6 and especially Smad7-overexpressing cells became very large and flat, in contrast to the compact piles of rounded, lipid-filled cells in control cultures (Fig 10 B). The Smad7-overexpressing cells were generally larger and flatter than the Smad6-expressing cells, and had a higher incidence of giant cells, which were about fivefold larger than the surrounding cells (Fig 10 D). The few adipocytes that did appear in the Smad6 or Smad7 cell cultures were very small in comparison to control adipocytes, and did not accumulate much lipid (Fig 10 B, arrow). In the presence of TGF-ß, all cell lines had a similar morphology, with the exception of the Smad6 or Smad7 cell cultures, which contained a few of the remnant larger cells (Fig 10 C), and appeared larger overall.
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To identify the domain of Smad7 that is required for its effects on adipogenesis, we evaluated the effects of expression of two different Smad7 mutants. The Smad7C mutant has a 19amino acid deletion at the COOH terminus, a mutation which abrogates the ability of Smad7 to block TGF-ß receptor signaling (
C expressing cells accumulated lipid similarly as the vector control cells, whereas lipid accumulation was blocked in the Smad7C expressing cells (Fig 11 B). Differentiation, as assessed by expression of PPAR
mRNA, also indicated that Smad7C, but not Smad7
C, decreased differentiation (Fig 11 C). Staining and PPAR
mRNA expression also revealed that Smad7C, but not Smad7
C, cooperated with TGF-ß in blocking differentiation (Fig 11B and Fig C). Smad7C, but not Smad7
C, also synergized with TGF-ß in stimulating DNA synthesis (Fig 11 D). Finally, the phenotype of the Smad7C cells resembled that of cells expressing wild-type Smad7, whereas Smad7
C-expressing cells resembled vector control cells (Fig 11 E). While Smad7C was qualitatively identical to wild-type Smad7 in our assays, it was quantitatively a weaker effector in comparison to full-length Smad7.
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Our results indicate that Smad6 and Smad7 enhance, rather than inhibit, the effects of TGF-ß signaling on adipogenic differentiation and cell proliferation. The C-domain of Smad7, which has been shown to be required for inhibition of TGF-ß signaling in other experimental systems, is necessary and sufficient for its effects on adipocytic differentiation in 3T3-F442A cells.
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Discussion |
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The ability of TGF-ß and TGF-ßrelated factors to regulate mesenchymal differentiation is well documented. Nevertheless, little is known about the role of endogenous signaling by TGF-ß, or its related factors, or the Smads, in cell differentiation. In this study, we have investigated the functions of endogenous TGF-ß and Smad signaling in adipocyte differentiation, using a well-known model system for adipocyte differentiation. While exogenous TGF-ß is known to inhibit adipocyte differentiation, TGF-ß also has been shown to be endogenously produced by adipocytes. Furthermore, preadipocytes secrete and activate TGF-ß, whereas mature adipocytes do not (
Regulation and the Role of TGF-ß Receptor Signaling in Adipogenesis
To characterize the potential role of autocrine TGF-ß in adipose differentiation, we assessed the availability of receptors for TGF-ß binding during differentiation. We found that the cell-surface expression of the TGF-ß receptors is strongly downregulated during adipose differentiation. Our results are in accordance with the strong decrease in ligand binding during differentiation of primary rat adipocytes (
The downregulation of cell-surface TGF-ß receptors in 3T3-F442A adipocytes is reminiscent of observations in other types of mesenchymal differentiation. Differentiation of myoblasts into myocytes is accompanied by a strong decrease in TGF-ß binding sites at the cell surface (
The differentiation-inhibiting role of autocrine TGF-ß in adipogenesis was confirmed in cells expressing a dominant negative version of the type II TGF-ß receptor. Even though we did not obtain high levels of dnTßRII expression, the cells showed a decreased response to TGF-ß. The dnTßRII cells had accelerated differentiation, suggesting that autocrine TGF-ß responsiveness regulates adipose conversion. These observations suggest that decreased receptor availability during adipogenesis allows these cells to escape the autocrine differentiation-inhibitory and growth-promoting roles of TGF-ß. The observation that retrovirally expressed cell-surface wild-type or dnTßRII were not decreased during differentiation, whereas endogenous cell-surface TßRII was decreased without a decrease in mRNA levels, suggests that the differentiation-dependent decrease in receptor availability results from translational repression.
Regulation and the Roles of Smad2 and Smad3 in Adipogenesis
Smad2 and Smad3 are both activated in response to TGF-ß and act, in cooperation with Smad4, as effectors of the TGF-ß response. All three Smads are expressed in 3T3-F442A cells, and their mRNA levels do not change significantly during differentiation. Whether Smad protein levels or their activation are altered during differentiation is currently unknown and awaits the availability of better antibodies. Our data also show that TGF-ß represses Smad3 but not Smad2 mRNA expression, and that this TGF-ßinduced repression decreases with differentiation, which is consistent with the downregulation of the cell-surface TGF-ß receptors. Whether cells alter Smad expression and activation as a mechanism to regulate TGF-ß responsiveness during differentiation is currently unclear. However, a recent report suggests differential expression of Smads at different stages during maturation of chondrocytes (
To characterize the roles of Smad2 and Smad3, we generated 3T3-F442A cells that stably overexpress either wild-type or dominant negative versions of Smad2 or Smad3. We assessed the effect of these manipulations on preadipocyte growth and differentiation, and the TGF-ß response. This study revealed different roles for Smad2 and Smad3. Overexpression of Smad3 decreased differentiation and increased proliferation in the absence or presence of TGF-ß, suggesting that Smad3 overexpression mimicked and enhanced the TGF-ß response. Conversely, dominant negative interference with Smad3 signaling enhanced the rate and extent of differentiation and inhibited cell proliferation. These results suggest that Smad3 mediates the differentiation-inhibiting and proliferative responses to TGF-ß.
In contrast to Smad3, alterations of Smad2 function did not affect cell proliferation, in the absence or presence of TGF-ß. However, increased Smad2 levels inhibited differentiation, albeit to a milder extent than Smad3, and enhanced the differentiation inhibitory response to TGF-ß. Remarkably, expression of dominant negative Smad2 had a similar, but smaller, effect on differentiation as wild-type Smad2. These observations were not peculiar to the SSXS mutations, since cells stably expressing dominant negative versions of Smad2 or Smad3 with the last three serines mutated to alanines (
SSXS mutants (data not shown). In principle, this effect could be explained by a possible weak residual activity of the mutated Smad2
SSMS or Smad2SA; however, this seems unlikely since we did not observe such behavior with the corresponding mutants of Smad3. Since Smad2 is also activated by activin, dominant negative interference with Smad2 signaling may primarily inhibit activin signaling, and this effect could explain the ability of Smad2
SSMS to inhibit differentiation and to enhance the TGF-ß response. Whether activin signaling contributes to adipogenesis remains to be explored since nothing is yet known about the function of activin in adipocyte differentiation.
Regulation and the Roles of Smad6 and Smad7 in Adipogenesis
While Smad2 and Smad3 are effectors of the TGF-ß response, Smad6 and Smad7 have been shown to act as inhibitors of TGF-ß responses. In contrast to Smad2 and Smad3, Smad6 and Smad7 mRNA expression is strongly repressed as adipogenesis proceeds. Since TGF-ß can induce Smad6 and Smad7 expression (
The downregulation of Smad6 and Smad7 suggested that these Smads normally exert an inhibitory effect on adipocyte differentiation, which is suppressed by the cells to allow full differentiation. Alternatively, the inhibitory effect of Smad6 and Smad7 on the TGF-ß response could suggest that increased Smad6 or Smad7 expression would favor adipogenic differentiation. Remarkably, Smad6 or Smad7 overexpression blocked adipogenesis and enhanced the inhibitory effect of TGF-ß on differentiation. In addition, Smad6 or Smad7 overexpression also enhanced the TGF-ßinduced stimulation of cell proliferation, without changing the growth rate in the absence of exogenous TGF-ß. This effect contrasts with the growth stimulatory effect of Smad3 overexpression, which enhances cell proliferation both in the absence or presence of TGF-ß. While the activities of Smad6 and Smad7 appeared qualitatively the same, Smad7 was more potent than Smad6, in spite of its lower level of overexpression.
The mechanisms by which Smad6 and Smad7 block adipocyte differentiation and enhance the TGF-ß response are unclear. However, we determined that the C-domain of Smad7 is necessary and sufficient for these effects in 3T3-F442A cells. This region is also required for the inhibitory effect of Smad7 on TGF-ß receptor signaling (
The molecular basis for the remarkable phenotype of the large cells in the Smad6 or Smad7 overexpressing cell lines also remains to be determined. It is conceivable that this phenotype results from an indiscriminate block in autocrine responsiveness to the different TGF-ßrelated factors by Smad6 or Smad7. This blockage may disable the mesenchymal differentiation program, which would be consistent with the ability of Smad7 to block mesoderm differentiation and to induce formation of neural tissue in Xenopus (
In summary, our results suggest that autocrine TGF-ß regulates the rate of adipose conversion, and that cells reduce their autocrine response to TGF-ß by repressing the availability of the TGF-ß receptors as they differentiate. The effects of TGF-ß on preadipocyte differentiation are mediated by Smad2 and Smad3, which have distinct functions in differentiation. The mechanisms by which Smad2 and Smad3 block differentiation may be related to their well-known ability to interact with and modify the activity of transcription factors. Whether these Smads target the transcription factors that drive adipose differentiation is currently under investigation. Since TGF-ß did not affect the induction of C/EBPß and -, but blocked the induction of ADD-1, PPAR
, and C/EBP
, Smad2 or Smad3 may exert their effects at some point upstream of ADD-1 and PPAR
induction. The function of Smad6 and Smad7 may be to block premature differentiation (akin to the function of autocrine TGF-ß production), possibly by blocking several TGF-ß family signaling pathways. The current observations provide a basis for the investigation of the molecular mechanisms whereby these Smads regulate adipocyte differentiation.
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Footnotes |
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1 Abbreviations used in this paper: dnTßRI and II, type I and II TGF-ß receptor, respectively.
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Acknowledgements |
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We thank the following investigators for plasmids and cell lines: K. Easley (Harvard Medical School, Boston, MA), H. Green, S. Itoh, S. McKnight, K. Miyazono, G. Nolan, B. Spiegelman, P. ten Dijke, and X.-F. Wang (Duke University Medical School, Durham, NC). We also thank Y. Zhang for providing the Smad3SSVS mutant construct, and X.-H. Feng for rounding up many other constructs from outside investigators. We are grateful to E. Filvaroff and M. Bauzon (both from University of California, San Francisco, San Francisco, CA) for help with obtaining the retroviral packaging cell lines, and to T. Alliston, J. Qing, and H. Fan for helpful discussions and encouragement.
This research was supported by a grant from the Arthritis Foundation and the National Institutes of Health grants P50-DE10306 (Project IV) and P60-DE1305 (Project III) to R. Derynck, and postdoctoral fellowships from the American Cancer Society and the American Heart Association to L. Choy.
Submitted: 21 September 1999
Revised: 17 February 2000
Accepted: 14 March 2000
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References |
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