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Endocrinology Vol. 143, No. 7 2478-2485
Copyright © 2002 by The Endocrine Society

INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS

Down-Regulation of Aortic Carboxypeptidase-Like Protein during the Early Phase of 3T3-L1 Adipogenesis

AnneMarie Gagnon1, Kayvan J. Abaiian1, Thet Crapper, Matthew D. Layne and Alexander Sorisky

Departments of Medicine and Biochemistry, Microbiology, and Immunology, Ottawa Health Research Institute, University of Ottawa (A.G., K.J.A., T.C., A.S.), Ottawa, Canada K1Y 4E9; and Cardiovascular and Pulmonary Divisions, Brigham and Women’s Hospital and Harvard Medical School (M.D.L.), Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Alexander Sorisky, Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: . asorisky{at}ohri.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aortic carboxypeptidase-like protein (ACLP) is a 175-kDa protein that is expressed in vascular smooth muscle cells and contains a signal peptide sequence, a lysine- and proline-rich repeating motif, a discoidin-like domain with 35% identity to discoidin I, and a carboxypeptidase-like domain that is 39% identical with carboxypeptidase E. It is secreted into the extracellular matrix and may play a role in abdominal wall development and dermal wound healing. ACLP is also expressed in adipose tissue, but at lower levels. In this study we demonstrate that ACLP protein and mRNA are severely down-regulated in the early phase of 3T3-L1 preadipocyte differentiation induced by insulin, dexamethasone, and isobutylmethylxanthine. Neither dexamethasone, isobutylmethylxanthine, nor insulin treatment alone reduced the level of ACLP protein, suggesting that ACLP down-regulation is a differentiation-associated event. ACLP down-regulation coincided with the onset of the postconfluent mitotic clonal expansion phase of adipogenesis. In contrast, subconfluent 3T3-L1 cell proliferation did not alter ACLP expression, suggesting a specific linkage between ACLP and differentiation-induced clonal expansion. Stable overexpression of ACLP had no effect on preadipocyte differentiation assessed by triacylglycerol accumulation and peroxisome proliferator-activated receptor-{gamma} levels. The role of ACLP and its marked reduction during adipogenesis merit further study.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POSITIVE ENERGY BALANCE leads to increased adipose tissue mass through augmentation of both adipose cell size and number (1). New adipocytes are formed from the differentiation of fibroblast-like precursors (pre-adipocytes) residing in the stromal-vascular fraction of adipose tissue (2). The murine 3T3-L1 preadipocyte cell line has been extensively used to study cellular and molecular pathways involved in preadipocyte differentiation and serves as a model of the in vivo process of adipogenesis (3, 4, 5). Confluent, growth-arrested 3T3-L1 preadipocytes are induced to differentiate with insulin in the presence of dexamethasone and isobutylmethylxanthine (IBMX) for the first 2 d. Differentiation is first characterized by reentry into the cell cycle and one or two rounds of postconfluent mitoses, termed clonal expansion (6, 7), which allows the de-repression of genes required for adipogenesis and permits amplification of committed cells. Induction of the transcription factors peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and CCAAT/enhancer-binding protein-{alpha} (C/EBP{alpha}) precedes and may regulate the second and permanent exit from the cell cycle (8, 9). The terminal differentiation stage is defined by acquisition of the mature lipid-filled rounded adipocyte phenotype (5, 6, 7, 10).

During differentiation, preadipocytes undergo significant morphological changes, due to important alterations in their cytoskeleton and extracellular matrix (ECM) (6). Levels of the cytoskeletal proteins actin and tubulin as well as production of collagen I/III and fibronectin are markedly decreased upon differentiation of 3T3-L1 preadipocytes (11, 12, 13). In contrast, the expression of collagen type IV and entactin is increased (14). Increases in matrix deposition, through either exposure to TGFß or growth on fibronectin-coated plates, suppresses 3T3-L1 preadipocyte differentiation and attenuates adipogenic insulin signaling (15, 16, 17). Similarly, the pre-adipocyte factor 1 may inhibit differentiation by stabilizing the matrix-cytoskeleton interaction through its epidermal growth factor-like repeats, which are found in many ECM proteins and cell adhesion molecules (18).

Aortic carboxypeptidase-like protein (ACLP) is a secreted 175-kDa protein that was initially cloned from aortic vascular smooth muscle cells, where it was shown to be up-regulated during differentiation (19). ACLP contains a signal peptide sequence, a proline- and lysine-rich repeating motif, a discoidin-like domain, and a C-terminal domain that is 39% identical with carboxypeptidase (CP) E. Despite this homology, ACLP is enzymatically inactive (19, 20). It is located in the ECM of collagenous tissues, where it plays a role in embryonic development and dermal wound healing (21). ACLP is also expressed in adipose tissue, but at lower levels (21). Its carboxyl terminus is identical with adipocyte enhancer binding protein-1 (AEBP-1) (22), which appears to be a truncated clone of ACLP (19). Given its potential role in ECM interactions, we investigated the regulation and function of ACLP in 3T3-L1 adipogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preadipocyte cultures and preadipocyte differentiation
The 3T3-L1 preadipocyte cell line was obtained from American Type Culture Collection (Manassas, VA), and the 3T3-C2 and 3T3-F442A cell lines were provided by Dr. Howard Green (Harvard University, Boston, MA) (23). The cells were grown to confluence in standard growth medium consisting of DMEM supplemented with 10% calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 C in a 10% CO2 atmosphere. Differentiation was induced on d 0 (2-d postconfluent cells) by addition of 100 nM insulin, 0.5 mM IBMX, and 0.25 µM dexamethasone. After 48 h (d 2), the medium was replaced with DMEM supplemented with 10% calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 100 nM insulin and was changed every 2 d up to d 8.

For experiments involving exposure of preadipocytes to individual components of the differentiation medium, growth-arrested d 0 (2 d postconfluence) cells were exposed to standard growth medium containing dexamethasone, IBMX, or insulin, alone or in combination (normal differentiation), at the above-mentioned concentrations. As in the standard differentiation protocol, after 48 h of exposure to dexamethasone or IBMX, cells were switched into growth medium only and cultured for another 2 d, whereas those exposed to insulin alone were kept in this medium (growth medium plus insulin) for the entire 4 d. For analysis of ACLP protein expression during subconfluent 3T3-L1 pre-adipocyte proliferation, cells were grown in standard growth medium and harvested at 60% confluence.

3T3-F442A preadipocytes were grown to confluence in DMEM supplemented with 10% calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin. Two days postconfluence (d 0) cells were induced to differentiate in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 1 µM insulin. Medium was changed every 2 d for an 8-d differentiation period. Mouse white adipocyte homogenate was a gift from Drs. A. Melnyk and J. Himms-Hagen (University of Ottawa, Ottawa, Canada).

Immunoblot analysis
At the specified time points during the 8-d differentiation protocol, or during subconfluent proliferation when indicated, cultured cell monolayers were lysed in Laemmli lysis buffer (24). Equal amounts of solubilized cellular protein (ranging from 60–150 µg depending on the experiment) were resolved by 7.5% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Nonspecific antigenic sites were blocked, and membranes were incubated with rabbit polyclonal anti-ACLP serum (1:1000) that was generated against the mouse ACLP fragment encoding amino acids 615-1128 (19), followed by incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL). Immunoreactivity was detected by enhanced chemiluminescence (NEN Life Science Products, Boston, MA). Where indicated, immunoblots were stripped of antibody and reprobed with mouse PPAR{gamma} antibody (1 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoblot exposures were scanned using the Gel Doc 1000 System (Bio-Rad Laboratories, Inc.), and bands were quantified by volume analysis using Molecular Analyst version 1.4 software (Bio-Rad Laboratories, Inc.).

Northern blot analysis
At specified time points during the differentiation protocol, RNA from cultured cells was isolated by guanidinium isothiocyanate extraction. Equal amounts of RNA samples (ranging from 2–4 µg) were electrophoresed on a 1% agarose gel containing 3% formaldehyde. RNA was transferred onto a Hybond-N nylon membrane (Amersham Pharmacia Biotech) in 3 M NaCl and 0.3 M trisodium citrate, pH 7. Equal loading and adequate transfer efficiency of RNA were verified by ethidium bromide staining of 28S and 18S rRNAs on the gel and the membrane. The membrane was then baked and prehybridized with 1 mg/ml salmon sperm DNA. The membrane was probed with the 0.7-kb cDNA fragment obtained by a SacI restriction digest of the full size ACLP cDNA (19) that was radiolabeled by random priming (Amersham Pharmacia Biotech). After several washes, ranging from 0.1x standard saline citrate at 60 C to 2x standard saline citrate at 25 C, the ACLP mRNA bands were detected by exposure of membrane to Kodak X-AR film (Eastman Kodak Co., Rochester, NY) and/or to a phosphor screen followed by scanning of the screen by a Typhoon 8600 Variable Mode Imager (Amersham Pharmacia Biotech).

Cell enumeration
3T3-L1 and 3T3-C2 cells were trypsinized on d 0, 2, 4, 6, and 8 after adipogenic stimulation and counted in duplicate using a Neubauer hemocytometer. Results represent the mean ± SEM of three independent experiments, each performed in duplicate.

Retroviral infections
The 293T/17-derived Phoenix-Eco packaging cells (American Type Culture Collection) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Upon reaching 80% confluence, cells were transiently transfected with pLXSN, pLXSN-ACLP, or no DNA (sham), by the calcium phosphate method. Viral supernatants were collected 48 h posttransfection, filtered, and applied in the presence of 4 µg/ml polybrene to exponentially growing 3T3-L1 preadipocytes for 24 h (25). Preadipocytes were grown to confluence and induced to differentiate as described above.

Determination of triacylglycerol (TG) mass
At the end of the 8-d differentiation protocol, cellular TG was extracted with isopropanol/heptane (2:3). Lipids were saponified, and TG mass was measured spectrophotometrically as previously described (26, 27). Cellular protein was assayed by a modified Lowry method, using BSA as standard.

Statistical analysis
Data were analyzed by ANOVA using InStat software (version 3.00, GraphPad Software, Inc., San Diego, CA) for Windows 95.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ACLP expression during 3T3-L1 preadipocyte differentiation
We analyzed the expression of ACLP protein during the 8-d process of 3T3-L1 preadipocyte differentiation. Induction of differentiation caused a marked decrease in ACLP protein expression (Fig. 1AGo). Within 2 d of adipogenic stimulation, ACLP protein expression decreased to 5 ± 2% (mean ± SEM; n = 4) of the level in d 0 cells. ACLP expression then slowly started to rise again, reaching 68 ± 9% of d 0 levels (mean ± SEM; n = 5) by d 8. ACLP expression remained constant in 3T3-L1 preadipocytes maintained for up to 8 d in growth medium lacking adipogenic agents (data not shown). ACLP is also expressed in two additional adipocyte models, the differentiated 3T3-F442A and primary mouse adipocytes (Fig. 2Go). Therefore, ACLP may play a role in adipogenesis.



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  		Figure 1. ACLP expression is down-regulated during early 3T3-L1 preadipocyte differentiation. Confluent 3T3-L1 preadipocytes were induced to differentiate as described. A and B, Equal amounts of solubilized protein were separated by SDS-PAGE, transferred, and immunoblotted with anti-ACLP or anti-PPAR{gamma} antibody. Representative blots are shown. Densitometric data are expressed as a percentage of the d 0 level and represent the mean ± SEM of three to five independent experiments, each performed in duplicate. Statistical analysis was performed using ANOVA. a, P < 0.001 vs. d 0; b, P < 0.001 vs. d 4, 6, and 8; c, P < 0.01 vs. d 6; d, P < 0.05 vs. d 0; e, P < 0.01 vs. d 1 and 3; f, P < 0.01 vs. d 0 and 1, and P < 0.001 vs. d 2 and 3. C, RNA was isolated and subjected to Northern blot analysis (4 µg/lane). The blots were hybridized with 32P-labeled ACLP cDNA probe. The gel was also stained with ethidium bromide to assess loading. Densitometric data are expressed as a percentage of the d 0 value and represent the mean ± SEM of three to five independent experiments, each performed in duplicate. Statistical analysis was performed using ANOVA. a, P < 0.001 vs. d 0 and 8; b, P < 0.001 vs. d 0, and P < 0.05 vs. d 8; c, P < 0.01 vs. d 0.

 


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  		Figure 2. ACLP is expressed in differentiated 3T3-F442A and mouse adipocytes. Confluent 3T3-F442A preadipocytes were induced to differentiate as described in the text. Lysates from mouse adipose tissue were obtained from Dr. Himms-Hagen’s laboratory. Equal amounts (25 µg) of solubilized proteins were separated by SDS-PAGE, transferred, and immunoblotted with anti-ACLP as described in the text.

 
To more precisely characterize the temporal decrease in ACLP protein levels, we measured ACLP protein expression every 24 h between d 0 and 4 of the differentiation protocol (Fig. 1BGo). The ACLP protein level declined to 41 ± 3% (mean ± SEM; n = 4) of that in d 0 cells within 24 h of exposure to the adipogenic stimulus and reached a maximal reduction to 9 ± 2% of that in d 0 cells (mean ± SEM; n = 4) by d 2. ACLP levels then progressively rose on d 3 and 4. Confirmation that the 3T3-L1 cells were differentiating under these conditions is provided by the expected up-regulation of PPAR{gamma}.

To gain further insight into the mechanism underlying the decrease in ACLP protein expression, Northern analysis was performed on RNA isolated from the cells at various times throughout the 8-d differentiation protocol. Figure 1CGo reveals that the decrease in ACLP protein expression was paralleled by a decrease in ACLP mRNA levels. This reduction in ACLP mRNA was not observed in cultures treated with control growth medium. ACLP mRNA was maximally inhibited (11 ± 7% of d 0 levels; mean ± SEM; n = 5) within 2 d of induction of differentiation. ACLP mRNA gradually increased by d 4. The general pattern of ACLP mRNA expression during adipogenesis is consistent with that seen for ACLP protein expression and suggests a transcriptional component to the differentiation-associated regulation of ACLP.

ACLP expression during the adipogenic stimulation of 3T3-C2 cells
To determine whether the ACLP down-regulation that is observed in 3T3-L1 cells is linked to terminal preadipocyte differentiation, we analyzed ACLP protein expression in the related 3T3-C2 cells exposed to the adipogenic cocktail that induces the differentiation of 3T3-L1 preadipocytes. The 3T3-C2 cells are also capable of entering the early clonal expansion phase of adipogenesis, but they are unable to progress through terminal differentiation, as they do not express PPAR{gamma} and do not accumulate lipid (28). The impaired adipogenic response of 3T3-C2 cells is related to a defect in PPAR{delta} induction; when engineered to express PPAR{delta}, they do differentiate fully (29). Interestingly, we observed a pattern of ACLP protein expression similar to that seen in differentiating 3T3-L1 cells. However, the extent of ACLP protein reduction was less pronounced for the 3T3-C2 cells. ACLP protein expression was transiently decreased to a minimum of 33 ± 8% (mean ± SEM; n = 3) of d 0 levels by d 2 of adipogenic stimulation (Fig. 3AGo). ACLP protein expression reverts completely to d 0 levels by d 4. Similarly, ACLP mRNA is reduced within 48 h of treatment with the adipogenic cocktail (to 4.6 ± 1.7% of the d 0 level; mean ± range; n = 2) and is then gradually increased, reaching 71 ± 5% (mean ± range; n = 2) of d 0 levels by d 4 (Fig. 3BGo). These results indicate that ACLP reduction occurs in cells that only proceed through the early phase of adipogenesis, as assessed by the lack of lipid droplets and the absence of PPAR{gamma} expression (Fig. 3AGo).



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  		Figure 3. ACLP protein expression is reduced in 3T3-C2 cells stimulated with adipogenic hormones. Confluent 3T3-C2 cells were treated with dexamethasone, IBMX, and insulin as described in the text. A, Equal amounts of solubilized proteins were separated by SDS-PAGE, transferred, and immunoblotted with anti-ACLP or anti-PPAR{gamma} antibody. Representative blots are shown. Densitometric data are expressed as a percentage of the d 0 value and represent the mean ± SEM of three independent experiments, each performed in duplicate. Statistical analysis was performed using ANOVA. a, P < 0.05 vs. d 0 and 4; b, P < 0.01 vs. d 0 and 4. B, RNA was isolated and subjected to Northern blot analysis (4 µg/lane). The blots were hybridized with 32P-labeled ACLP cDNA probe. The gel was also stained with ethidium bromide to assess loading. Densitometric data are expressed as a percentage of the d 0 value and represent the mean ± range of two independent experiments.

 
The dexamethasone/IBMX/insulin combination is necessary for ACLP down-regulation
Optimal 3T3-L1 preadipocyte differentiation requires converging input from dexamethasone, IBMX, and insulin (10, 30). To determine whether ACLP down-regulation is a result of the combined effects of these agents or is due to an individual component, ACLP protein expression was evaluated upon exposure to the individual components of the differentiation stimulus. As shown in previous experiments, normal differentiation caused a significant decline in ACLP protein levels by d 2 (to 5 ± 1% of the d 0 level; mean ± SEM; n = 3) with a return to 64 ± 16% of the basal level (mean ± SEM; n = 3) by d 4 (Fig. 4Go, lanes 2 and 6). Although some variation was noted, neither dexamethasone, insulin, nor IBMX alone for 2 d significantly down-regulated ACLP expression in 3T3-L1 preadipocytes (Fig. 4Go). These results suggest that ACLP down-regulation is differentiation associated.



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  		Figure 4. The combination of dexamethasone, IBMX, and insulin is required for ACLP down-regulation in 3T3-L1 cells. Confluent 3T3-L1 preadipocytes were induced to differentiate with dexamethasone (D), IBMX (M), and insulin (I), either alone or in combination, as described. Equal amounts (40 µg) of solubilized proteins were separated by SDS-PAGE, transferred, and immunoblotted with anti-ACLP antibody. A, Representative blot of ACLP expression. B, Densitometric analysis of ACLP. Data represent the mean ± SEM of three independent experiments. Statistical analysis was performed by ANOVA. a, P < 0.05 vs. d 0.

 
ACLP is specifically down-regulated during the postconfluent mitoses of clonal expansion
We noted a temporal correlation between the decreases in ACLP mRNA and protein expression and the differentiation-induced clonal expansion phase of 3T3-L1 and 3T3-C2 cells. Cell count experiments were performed to compare the clonal expansion of these two cell lines to determine whether the extent of clonal expansion correlates with the observed decline in ACLP protein levels. As expected, 3T3-L1 cells approximately doubled in number by d 4 (76 ± 11% increase in cell number; mean ± SEM; n = 4) and maintained a constant cell population thereafter (Fig. 5AGo). As reported in other studies (28), 3T3-C2 cells stimulated with the adipogenic cocktail also entered a limited clonal expansion phase. Cell numbers increased by 39 ± 10% (mean ± SEM; n = 3) by d 2 of hormonal treatment (Fig. 5BGo). The more prominent clonal expansion phase observed in 3T3-L1 cells, compared with 3T3-C2 cells, may be linked to the steeper decline in ACLP protein expression occurring in differentiating 3T3-L1 cells.



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  		Figure 5. 3T3-L1 and 3T3-C2 cells enter a clonal expansion phase upon stimulation with the adipogenic hormones. Confluent 3T3-L1 (A) and 3T3-C2 (B) cells were treated with dexamethasone, IBMX, and insulin or kept in control medium as described. On the indicated days, cells were trypsinized and counted in duplicate. Results represent the mean ± SEM of four (3T3-L1) or three (3T3-C2) independent experiments. —-, Control medium; - - -, adipogenic medium.

 
Our results could suggest that a decrease in ACLP expression may be required for generalized cell division. Indeed, decreased ACLP expression has been observed in proliferating smooth muscle cells compared with quiescent cells (19). However, in the 3T3-L1 cell model, we found that ACLP protein expression is unchanged in subconfluent growing cells vs. growth-arrested confluent cells (Fig. 6Go). These results indicate that the reduction in ACLP expression observed upon adipogenic stimulation is specific to the specialized mitoses associated with clonal expansion and is not a general feature of proliferation.



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  		Figure 6. ACLP protein expression is similar in growth-arrested confluent and rapidly proliferating subconfluent 3T3-L1 preadipocytes. Equal amounts of solubilized protein from 60% confluent and quiescent confluent 3T3-L1 cells were separated by SDS-PAGE, transferred, and immunoblotted with anti-ACLP antibody. A, Representative blot of ACLP expression. B, Densitometric analysis of ACLP. Data are expressed as a percentage of the d 0 value and represent the mean ± range of two independent experiments, each performed in duplicate.

 
Sustained overexpression of ACLP does not inhibit adipogenesis
To determine whether the decrease in ACLP expression was absolutely required for adipogenesis, we engineered 3T3-L1 preadipocytes to overexpress ACLP in a sustained manner. 3T3-L1 preadipocytes infected with retrovirus containing the full-length ACLP cDNA (pLXSN-ACLP) expressed approximately 6-fold more ACLP protein than cells infected by viral particles with no insert (pLXSN). Unlike the differentiation-induced decrease in ACLP observed in pLXSN-infected cells, ACLP expression in pLXSN-ACLP-infected cells remained constant throughout the differentiation protocol (Fig. 7AGo). Continuous elevated expression of ACLP did not inhibit differentiation, as assessed by the induction of the transcription factor PPAR{gamma} and accumulation of TG (Fig. 7BGo). Protein concentrations were comparably increased during the differentiation of ACLP-overexpressing cells vs. controls, suggesting that clonal expansion was not compromised. Indeed, differentiation induced a similar rise in cell numbers (fold of basal) in both empty vector- and ACLP-infected cells (mean ± range, 2.13 ± 0.29 vs. 2.11 ± 0.37, respectively; n = 2).



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  		Figure 7. Sustained expression of ACLP does not prevent adipogenesis. 3T3-L1 preadipocytes were retrovirally infected as described in the text and induced to differentiate for up to 8 d. Equal amounts (40 µg) of solubilized proteins were separated by SDS-PAGE, transferred, and immunoblotted with anti-ACLP and anti-PPAR{gamma} antibodies. Two independent experiments were performed, and similar outcomes were obtained. A, Representative blot of ACLP expression. B, Representative blot of PPAR{gamma} induction. C, Alternatively, TG mass was measured at the end of 8 d of differentiation. Data are expressed as micrograms of TG per milligram of protein and represent the mean ± range of two independent experiments. , Differentiated cells; {square}, controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We observed a marked decrease in ACLP protein and mRNA levels during the early stage of 3T3-L1 preadipocyte differentiation. This reduction is differentiation associated, as it results from the combined effects of dexamethasone, IBMX, and insulin and coincides with clonal expansion. The presence of ACLP protein in two additional adipocyte cell culture models and in sc fat in vivo (21) further strengthens the view that changes in ACLP levels may be an important part of adipogenesis. However, sustained overexpression of ACLP is insufficient to prevent 3T3-L1 preadipocyte differentiation.

Adipogenesis is a complex sequence of events that culminates in the differentiation of fibroblast-like cells into specialized lipid-filled adipocytes. Early in the differentiation program, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle for one or two rounds of mitosis (5, 7). Such clonal expansion apparently modulates the accessibility of key regulatory elements in the promoter region of differentiation-inducing genes and allows the cells to permanently exit the cell cycle at a distinct stage (GD) (31). Both C/EBP{alpha} and PPAR{gamma} have been implicated in the regulation of this second final growth arrest (8, 9, 32, 33), which is characterized by the induction of several genes, including the growth arrest and DNA damage-inducible gene 45 (gadd45) and cyclin-dependent inhibitors (p21 and p27) (32, 34).

The anti-adipogenic potential of several agents, such as TNF{alpha} and Ara-C, has been linked to their ability to block mitotic clonal expansion, suggesting that this early event is necessary for successful terminal differentiation (35, 36). However, some evidence has challenged this view. Differentiation of 3T3-L1 cells was apparently achieved without DNA replication, although a late-onset clonal expansion phase was not ruled out (37). Human preadipocytes, differentiated in culture, also do not proceed through clonal expansion (38, 39). It has been postulated that these cells may be at a more advanced, postclonal expansion stage of the differentiation program. DNA replication precedes the appearance of lipid droplets and expression of adipocyte-specific enzymes in vivo (40, 41), supporting the requirement of clonal expansion for preadipocyte differentiation.

Down-regulation of ACLP coincides with the start of clonal expansion in differentiating 3T3-L1 preadipocytes and 3T3-C2 cells stimulated with the adipogenic cocktail. Members of the C/EBP family of transcription factors and retinoblastoma (Rb)-like proteins (Rb, p107, and p130) have been implicated in regulating the specialized postconfluent mitoses of adipogenic clonal expansion leading to permanent exit from the cell cycle and commitment to differentiate (8, 28, 32, 42). Furthermore, the Rb-like protein family modulates the DNA-binding activity of E2F transcription factors, key regulators of the cell cycle and differentiation (43). During adipogenesis, clonal expansion is associated with a switch in the E2F binding complex, from p130/E2F to p107/E2F, which is reversed upon permanent exit from the cell cycle (28). As with ACLP down-regulation, the p130 to p107 switch is also observed during the clonal expansion of hormonally stimulated 3T3-C2 cells, is specific to the differentiation-dependent reentry into the cell cycle, and is not observed in subconfluent mitoses (28). Our data suggest that ACLP down-regulation may be another distinct feature of the specialized mitoses of clonal expansion associated with adipogenesis, but not subconfluent proliferation. Further studies are required to implicate ACLP in the regulation of clonal expansion.

The function of ACLP is still unclear. There is no overt adipose-specific aberration in mice in which the ACLP gene has been deleted (Layne, M. D., unpublished observations). However, the gastroschisis phenotype of the ACLP-/- mouse is similar to that observed in the knockout model of the bone morphogenic protein 1, a procollagen C proteinase (44). Further evidence also points to a role for ACLP in collagen fiber formation or stabilization. ACLP is secreted into the ECM of collagen-rich tissues including the vasculature, dermis, and the developing skeleton where it contributes to embryonic development and dermal wound healing (21). This may involve its N-terminal domain with homology to discoidin I (19), a slime mold protein that functions in cell aggregation (45). Several mammalian proteins carrying such discoidin-like domains have been identified, including the discoidin-like domain receptors (46). These tyrosine kinase receptors are activated by various types of collagens via specific binding sites in their discoidin-like domain (47).

Early adipocyte differentiation is also characterized by complex changes in the levels and types of ECM components (6). Although this event has not received as much attention as clonal expansion, destabilization of the ECM and cytoskeleton early in the differentiation program is critical for terminal differentiation. Treatment of cells with TGFß (36) or culture onto matrix-coated plates strongly inhibits adipocyte differentiation in 3T3-L1 and 3T3-F442A cells (15, 16, 17). Collagen type IV induction is observed at confluence and continues to rise upon adipogenic stimulation (14). It is accompanied by a decrease in collagen types I and III and fibronectin production (12, 13). Reduction in polymerized actin and tubulin is also observed and is thought to permit the drastic cellular morphological changes required for the conversion of a flat, fibroblastic cell to a rounded, lipid-filled adipocyte (11). We report here that another ECM component, ACLP, is reduced at the onset of adipogenesis, concomitantly with decreases in collagen types I and III. However, despite the potential role of ACLP in the formation/stabilization of collagen fibers, its overexpression appears not to prevent proadipogenic effects of the severe decreases in collagen types I and III. This could explain the lack of effect of ACLP overexpression we observed on 3T3-L1 preadipocyte differentiation.

The discoidin-like domain/CP structure of ACLP is found in two other proteins, CPX-1 and CPX-2, which are also secreted by mammalian cells (48, 49). Despite homology with CPE, these proteins are apparently not involved in protein processing because their CP domains lack key residues involved in enzymatic activity and have been shown to be inactive toward common CP substrates (20, 48, 49). Instead, ACLP, CPX-1, and CPX-2 have been postulated to act as binding proteins. Therefore, ACLP may act as a bridging protein, stabilizing the ECM by binding to collagen, via its discoidin-like domain, and another factor/protein, through its inactive CP domain.

Our results indicate that changes in ACLP mRNA levels occur during 3T3-L1 preadipocyte differentiation, which could be due to either transcriptional or posttranscriptional events, such as those affecting mRNA stability. Translational regulation of ACLP mRNA has also been suggested in the kidney (19) and may modulate ACLP expression during adipogenesis. Sequence analysis revealed that the C terminus of mouse ACLP is identical with AEBP-1, previously cloned as a binding protein for the adipocyte enhancer 1 (AE-1) element controlling the expression of the adipocyte-specific aP2 gene (22). AEBP-1 and ACLP cDNAs detect a single 4-kb mRNA product. AEBP-1 appears to be a partial clone of ACLP (19). Moreover, a longer version of AEBP-1, containing a full discoidin-like domain, was independently cloned in human primary osteoblasts (50). Furthermore, although the antibody used in our studies was generated against the common C-terminal domain of ACLP/AEBP-1, we were unable to detect the reported 80-kDa AEBP-1 protein. The pattern of ACLP mRNA expression observed in our studies is in agreement with previous studies demonstrating a decrease in AEBP-1 mRNA in differentiated 3T3-L1 cells vs. preadipocytes (22), and these results were recently confirmed independently through gene array analysis (51).

In summary, we describe the severe down-regulation of ACLP, an ECM component, during the early phase of adipocyte differentiation. The decline in ACLP occurs concurrently with the onset of clonal expansion and the destabilization of the ECM. These two events are critical for the adipogenic differentiation program and suggest that ACLP is a participant in the early phase of adipogenesis. Further characterization of the function of ACLP is required to advance our knowledge of preadipocyte differentiation and adipose tissue physiology.


   Footnotes
 
This work was supported by a grant from the Physicians’ Services Inc. Foundation.

1 A.G. and K.J.A. contributed equally to this paper. Back

K.J.A. was supported by an Ontario Graduate Studies Scholarship. A.G. is the recipient of a Premier’s Research Excellence Award held by A.S. A.S. is a Career Investigator with the Heart and Stroke Foundation of Ontario.

Abbreviations: ACLP, Aortic carboxypeptidase-like protein; AE-1, adipocyte enhancer 1; AEBP-1, adipocyte enhancer-binding protein-1; C/EBP, CCAAT/enhancer-binding protein; CP, carboxypeptidase; CPE, carboxypeptidase E; CPX, carboxypeptidase X; ECM, extracellular matrix; IBMX, isobutylmethylxanthine; PPAR{gamma}, peroxisome proliferator-activated receptor-{gamma}; Rb, retinoblastoma; TG, triacylglycerol.

Received July 7, 2001.

Accepted for publication March 4, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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