Original Article Differential Expression of Matrix 3 (MMP3) in Preadipocytes/Stromal Vascular Cells From Nonobese Nondiabetic Versus Obese Nondiabetic Pima Indians Michael T. Traurig, Paska A. Permana, Saraswathy Nair, Sayuko Kobes, Clifton Bogardus, and Leslie J. Baier

Prior microarray studies comparing global expression vascular cells) isolated from nonobese and obese subjects patterns in preadipocytes/stromal vascular cells isolated and identified 218 that were differentially expressed from nonobese nondiabetic versus obese nondiabetic Pima between these two groups. One of the genes most notably Indians showed that 9 (MMP9)is upregulated in preadipocytes/stromal vascular cells iso- upregulated in obese subjects. The current study targeted lated from obese subjects was MMP9, and this upregula- analysis of nine additional MMP genes that cluster to a tion was confirmed by real-time PCR (3). Matrix region on 11q22 that is linked to BMI and metalloproteinase 9 (MMP9) is a member of a large family percent body fat. Differential-display PCR showed that of MMPs, which have been proposed to be involved in MMP3 is downregulated in preadipocytes/stromal vascular cells from obese subjects, and real-time PCR showed that adipose tissue remodeling (4,5). Therefore, other MMP MMP3 expression levels are negatively correlated with family members, in addition to MMP9, may potentially percent body fat. To determine whether variants within contribute to the etiology of obesity (4,5). MMP3 are responsible for its altered expression, MMP3 A cluster of nine additional MMP genes (MMP7, MMP20, was sequenced, and seven representative variants were MMP27, MMP8, MMP10, MMP1, MMP3, MMP12, and genotyped in 1,037 Pima subjects for association analyses. MMP13) maps within a ϳ500-kb region located at chro- Two variants were associated with both BMI and type 2 mosome 11q22. In the current study, this cluster of MMP diabetes, and two additional variants were associated with genes at chromosome 11q22 was targeted for analysis type 2 diabetes alone; however, none of these variants were because this chromosomal region has previously been associated with MMP3 expression levels. We propose that the MMP3 pathway is altered in human obesity, but this linked to measures of obesity. Linkage to BMI at chromo- alteration may be the result of a combination of genetic some 11q22 has been reported in studies of Nigerian variation within the MMP3 locus itself, as well as variation families (6) and Old Order Amish (7), and linkage to BMI in additional factors, either primary or secondary to obe- at 11q22-25 has also been reported in a Dutch cohort of sity, that regulate expression of the MMP3 gene. Diabetes type 2 diabetic patients (8). Our group has also reported 55:3160–3165, 2006 linkage to percent body fat at chromosome 11q22 in a study of 277 Pima-Indian siblings (9), and, in a larger study of 966 Pima-Indian siblings, we reported linkage to BMI and type 2 diabetes at a more telomeric region (11q23-24) he Pima Indians of Arizona have a high preva- on (10). Among the nine MMP genes that lence of obesity and type 2 diabetes, and both of map to chromosome 11q22, only MMP3 was detected in these diseases have a strong genetic basis (1,2). preadipocytes/stromal vascular cells isolated from Pima TTo identify novel genes and/or genetic pathways Indians. This gene was further analyzed using the tech- that may be important for the development of obesity in niques of differential-display PCR, real-time PCR, direct this Native-American population, Nair et al. (3) compared sequencing, and association analyses for obesity and type Affymetrix oligonucleotide microarray expression profiles 2 diabetes. from adipocyte precursor cells (preadipocytes/stromal

RESEARCH DESIGN AND METHODS From the Diabetes Molecular Genetics Section, Phoenix Epidemiology and Subjects were participants of ongoing longitudinal studies of the etiology of Clinical Research Branch, National Institute of Diabetes and Digestive and type 2 diabetes among the Gila River Indian Community in Arizona (11). Kidney Diseases, National Institutes of Health, Phoenix, Arizona. Subjects for the adipose tissue biopsy were admitted as inpatients into our Address correspondence and reprint requests to Leslie J. Baier, PhD, NIDDK, NIH, 445 North 5th St., Phoenix, AZ 85004. E-mail: [email protected]. Clinical Research Center and were confirmed to be nondiabetic, as deter- nih.gov. mined by an oral glucose tolerance test; the results of which were interpreted Received for publication 21 March 2006 and accepted in revised form 31 according to World Health Organization criteria (12). Body composition was July 2006. estimated by underwater weighing until January 1996, and thereafter it was GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metal- estimated by dual-energy X-ray absorptiometry (DPX-1; Lunar Radiation, loproteinase; SNP, single nucleotide polymorphism; UTR, untranslated region. Madison, WI). A conversion equation derived from comparative analyses was DOI: 10.2337/db06-0373 used to make the estimates of body composition equivalent between the two © 2006 by the American Diabetes Association. methods (9). For the differential-display PCR experiments, 32 nondiabetic 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 full-blooded Pima-Indian subjects were studied. None of these subjects were with 18 U.S.C. Section 1734 solely to indicate this fact. first-degree relatives. Of these 32 subjects, 16 were nonobese and 16 obese,

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TABLE 1 Subjects used for the differential-display PCR and real-time PCR n Age (years) BMI (kg/m2) Percent body fat Differential-display PCR Lean men 8 31 (21–38) 25 (20–29) 22 (15–29) Lean women 8 33 (26–36) 24 (19–30) 30 (20–35) Obese men 8 29 (21–37) 54 (46–70) 37 (33–41) Obese women 8 33 (27–48) 53 (47–66) 46 (45–48) Real-time PCR Total men 21 29 (18–43) 39 (20–74) 30 (11–47) Total women 26 31 (18–43) 40 (21–70) 40 (28–48) Data are means (range), unless otherwise indicated.

and they were pair matched for age and sex (Table 1). For real-time PCR, 47 cDNA was synthesized from preadipocytes/stromal vascular cell total RNA, nondiabetic full-blooded Pima subjects (BMI range 20–70 kg/m2) were studied using a BD Advantage RT-for-PCR kit (BD Bioscience/Clontech, Palo Alto, (Table 1), of which 7 of these subjects had been previously studied for MMP9 CA), following the manufacturer’s instructions. expression. Sequencing of the MMP3 gene was performed on DNA from 18 (9 Differential-display PCR for the nine MMP genes located at 11q22. PCR nonobese and 9 obese) subjects who had also been studied by differential- amplification of MMP7, MMP20, MMP27, MMP8, MMP10, MMP1, MMP3, display PCR. Variants detected in the MMP3 gene were genotyped in a MMP12, and MMP13 was performed using specific primers (sequences for the family-based sample of 1,037 (573 diabetic, 464 nondiabetic) Pima Indians for primers are available from the authors on request). The PCR mix consisted of association analysis. All studies were approved by the Tribal Council and the the following components: 1 ␮l first-strand cDNA, 1 ␮l each of forward and institutional review board of the National Institutes of Diabetes and Digestive reverse primers (20 ␮mol/l each), 2 ␮lof10ϫ cDNA PCR buffer, 0.2 ␮lof50ϫ and Kidney Diseases. dNTP mix, 0.4 ␮l Advantage cDNA polymerase mix (Advantage cDNA PCR kit; ␮ ␮ Isolation of preadipocytes/stromal vascular cells. Subjects for adipose BD Bioscience/Clontech), and 14.4 ldH2O for a total volume of 20 l. Each tissue biopsies were admitted as inpatients to our Clinical Research Center, of the cDNA samples was amplified in duplicate, and the products were run and, after an overnight fast, underwent a subcutaneous abdominal needle and visualized on 2% high-resolution agarose gels (Agarose SFR; AMRESCO, biopsy under local anesthesia with 1% lidocaine. digestion of the Solon, OH). subcutaneous abdominal adipose tissue biopsy samples was performed as Real-time quantitative PCR for MMP3. MMP3 expression was quantitated previously described (13,14). Briefly, the preadipocyte-containing infranatant by real-time PCR, using a predesigned assay (Assays-on- was collected into a separate tube and washed several times with Hanks’ Demand gene expression products; Applied Biosystems, Foster City, CA). balanced salt solution. The stromal vascular fraction pellet containing prea- Real-time PCR was performed using an ABI Ϫ7700 sequence detection system dipocytes/stromal vascular cells was resuspended in standard medium con- (Applied Biosystems), following the manufacturer’s instructions. Assays were sisting of Medium 199 (Life Technologies, Grand Island, NY) supplemented performed in triplicate, and the mean values were used to calculate expres- with 1 ␮g/ml amphotericin B, 100 units/ml penicillin G sodium, 100 ␮g/ml sion levels, using the relative standard curve method. Glyceraldehyde-3- streptomycin sulfate, 2 mmol/l Glutamax-1, and 10% heat-inactivated fetal phosphate dehydrogenase (GAPDH) was used as the endogenous control to bovine serum (Life Technologies). The suspension was strained through a obtain normalized values. Raw GAPDH levels were plotted against both BMI sterile 25-um stainless steel tissue sieve (Thermo EC, Holbrook, NY). The and percentage of body fat to establish that there was no relationship between filtrate was transferred to a T75 culture flask and maintained in an incubator the GAPDH housekeeping gene and these phenoytpes.

at 37°C in 5% CO2. Cells were allowed to attach, and the next day floating Sequencing of MMP3. Overlapping primers were designed to sequence erythrocytes were removed by aspiration and the culture media replenished. ϳ2,900 nucleotides upstream of the transcription start site, all 10 exons, all 9 At subconfluency, the cultured cells were trypsinized and plated at a concen- introns, and ϳ2,400 nucleotides of the 3Ј-untranslated region (UTR) of the tration of ϳ1.5 ϫ 106 cells per 15-cm dish for RNA extraction, which was MMP3 gene. Sequencing was performed on DNA samples from 18 of the carried out ϳ14 days after the biopsy date. Media was changed every 2–3 days subjects (9 nonobese and 9 obese) studied by differential-display PCR, using throughout the culturing period. There were no differences (in terms of cell a Big Dye Terminator v1.1 cycle sequencing kit (Applied Biosystems). number or visible morphology) between the cultures from the nonobese and Genotyping of sequence variations in MMP3. All variants were genotyped obese subjects. Cultures were routinely screened for expression levels of in 1,037 Pima Indians. The two insertion/deletions variants, Ϫ1613 5T/6T ECSM2 (endothelial cell–specific molecule 2), a corollary of endothelial (rs3025058) and ϩ1494 7T/8T, were genotyped by direct sequencing as described contamination, to confirm that it is consistent and low among subjects. above. The variant Ϫ1475 (G/A) was genotyped by SNaPshot minisequencing Isolation of total RNA and synthesis of cDNA. Total RNA was extracted (SNaPshot multiplex kit; Applied Biosystems), following the manufacturer’s from the preadipocytes/stromal vascular cells, using an RNeasy Mini Kit protocol. PCR-amplified samples were analyzed on an ABI Prism 3700 DNA (Qiagen, Valencia, CA). To remove any residual DNA, the purified RNA was analyzer with GeneScan analysis software (Applied Biosystems). treated with DNase using an RNase-free DNase set (Qiagen). First-strand All other polymorphisms were genotyped by allelic discrimination (either

FIG. 1. Representative agarose gels (three different nonobese and obese matched pairs) showing a differentially expressed band (decreased in obese subjects) corresponding to MMP3 (arrow). For differential-display PCR, the cDNA samples were amplified in duplicate using MMP3 mRNA–specific primers.

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tween age- and sex-matched pairs of nonobese versus obese subjects showed that in 7 of the 16 pairs, MMP3 expression was high in the nonobese subjects but barely detectable or undetectable in the matched obese subjects (Fig. 1). In the other nine pairs, MMP3 expression was high in both the nonobese and matched obese subjects. All of the nonobese subjects (both male and female subjects) had comparable levels of MMP3 expression. Among the seven obese subjects that had downregulated MMP3 ex- pression, four were men and three were women. The identity of the differentially expressed product was con- firmed to be MMP3 by sequencing. Real-time quantitative PCR for MMP3. To confirm and quantitate the observed decrease in expression of MMP3 in some obese subjects, MMP3 expression levels were FIG. 2. Relationship between MMP3 mRNA levels and percent body fat, further examined by real-time quantitative PCR, using a adjusted for age and sex. MMP3 gene expression levels were measured larger sample (n ϭ 47) of Pima Indians who had a range of by real-time quantitative PCR in preadipocytes/stromal vascular cells ϭ 2 isolated from 47 nondiabetic Pima Indians, representing a range in BMI values (mean BMI 40 kg/m , range 20–70). Assays percent body fat. Messenger RNA levels, measured in triplicate, were were performed in triplicate, and the averages were used normalized using GAPDH as the endogenous control, after first plot- to calculate expression levels, which were normalized to ting raw GAPDH levels against percent body fat to establish that there was no relationship between GAPDH and percent body fat in these 47 GAPDH as an endogenous control. Linear regression anal- -yses were performed to determine whether mRNA expres .(0.04 ؍ subjects (r sion levels were correlated with measures of obesity. Assays-on-Demand single nucleotide polymorphism [SNP] genotyping prod- MMP3 mRNA levels were negatively correlated with per- ucts or Assays-by-Design service; Applied Biosystems) and were scanned cent body fat (P ϭ 0.004, adjusted for age and sex) (Fig. 2). using an ABI-7700 sequence detection system (Applied Biosystems). Statistical analysis. Statistical evaluations of the data were performed using Identification of sequence variants in MMP3. To de- a statistical analysis system (SAS Institute, Cary, NC). For continuous termine whether the decrease in MMP3 expression in variables, the general estimating equation procedure was used to adjust for obese subjects is attributable to variation in the MMP3 appropriate covariates, including age and sex. General linear regression gene itself, or is the result of variation in a factor/pathway models were used to analyze the relationships between mRNA expression that regulates MMP3, the MMP3 gene was sequenced in levels and obesity (percent body fat). DNA from 18 of the subjects studied by differential-display PCR (9 nonobese and 9 obese). The entire gene was RESULTS sequenced (ϳ2,900 bp upstream of the transcription start Differential-display PCR of each of the nine MMP site, all 10 exons and 9 introns, and ϳ2,400 bp of the genes positioned at chromosome 11q22. Gene expres- 3Ј-UTR). sion levels for MMP7, MMP20, MMP27, MMP8, MMP10, Sequencing of MMP3 identified 24 variants (Fig. 3). MMP1, MMP3, MMP12, and MMP13 were analyzed by Among the eight variants found in the 5Ј-UTR, only the G/A differential-display PCR, using cDNA synthesized from transition at Ϫ1475 appears to be novel. Seven of the preadipocytes/stromal vascular cell total RNA isolated 5Ј-UTR variants were single– substitutions, and from 16 nonobese and 16 obese Pima subjects (Table 1). one variant (rs3025058, often reported in the literature as Among these nine MMP genes, only MMP3 expression was 5A/6A) was a thymine insertion/deletion in a string of detected in preadipocytes/stromal vascular cells using this thymines (5T/6T) at position Ϫ1613. Among the 11 vari- technique. Comparison of MMP3 expression levels be- ants identified in the coding and intronic regions, 1

,FIG. 3. Variants identified by sequencing the MMP3 gene. Regions that were sequenced include 2,900 bp of the 5؅-UTR, all 10 exons and 9 introns ,and 2,400 bp of the 3؅-UTR. *Base positions are relative to the major transcription start site (Homo sapiens MMP3 gene, complete sequence accession no. AF405705).

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TABLE 2 Linkage disequilibrium coefficients (DЈ) and ⌬2 (r2) between pairs of variants in the MMP3 gene rs3025058 Ϫ1475 rs522616 rs679620 rs3020919 ϩ1494 rs650108 rs3025058 — Ϫ0.78 0.98 0.98 Ϫ0.62 Ϫ1.00 1.00 Ϫ1475 (Ͻ0.01) — 0.96 Ϫ0.79 Ϫ1.00 Ϫ0.82 Ϫ0.84 rs522616 (0.42) (0.04) — 1.00 0.98 Ϫ1.00 1.00 rs679620 (0.96) (Ͻ0.01) (0.45) — Ϫ0.73 Ϫ1.00 1.00 rs3020919 (0.01) (Ͻ0.01) (0.11) (0.01) — Ϫ0.67 0.98 ϩ1494 (Ͻ0.01) (Ͻ0.001) (0.01) (Ͻ0.01) (Ͻ0.001) — Ϫ1.00 rs650108 (0.70) (0.01) (0.61) (0.70) (0.16) (Ͻ0.01) — DЈ (above the diagonal) is the disequilibrium coefficient for association between alleles at two loci expressed as a proportion of the maximum possible disequilibrium given the observed allele frequencies and direction of association. The coefficient is calculated such that a positive value represents an association between the more common alleles at each locus, whereas a negative value represents an association between the common allele at one locus and the less common allele at the other locus. ⌬2 (r2, below the diagonal) is a measure of concordance; high values indicate high linkage disequilibrium and similar frequencies.

(rs679620) predicts a nonsynonymous substitution ated with type 2 diabetes (P ϭ 0.005 and 0.02, respectively, (Lys45Glu) in exon 2, and 2 (rs602128 and rs520540) under a dominant model adjusted for age, sex, birth date, predict synonymous substitutions in exons 2 and 8, re- family membership, and Pima heritage) (Table 4), but they spectively. Two of the intronic variants are thymine inser- were not associated with BMI (Table 3). However, among tion/deletions at ϩ496 and ϩ1494. The ⌬Tatϩ496 the 47 subjects who had measurements of MMP3 expres- (rs11422799) is in a string of 11 thymines in intron 1, and sion levels by real-time PCR, there was no correlation the ϩTatϩ1494 (novel) is in a poly (T) tract located 37 bp between genotype and MMP3 expression for any of these downstream of the 3Ј splice site for exon 4. Among the five variants (data not shown). variants in the 3Ј-UTR, one is novel (⌬Tatϩ9505). Genotyping and association analyses of the MMP3 variants. Based on the genotypic information obtained DISCUSSION while sequencing the 18 subjects, several of the variants Previous Affymetrix oligonucleotide microarray expres- identified in MMP3 were found to be in high linkage sion profiling of cultured abdominal subcutaneous preadi- disequilibrium (DЈϾ0.98, r2 ϭ 1), and therefore all 24 pocytes/stromal vascular cells isolated from the adipose variants could be divided into seven genotypic groups. To tissue of 14 nonobese and 14 obese nondiabetic subjects examine whether any of these variants were associated identified MMP9 as being upregulated in obese subjects with obesity and/or type 2 diabetes, one representative (3). A total of 7 subjects from this prior study of MMP9 and SNP from each group was genotyped in 1,037 (573 diabet- 40 additional subjects were studied in the current report, ic/464 nondiabetic) Pima Indians for association analyses. which shows that another member of the MMP family, None of the genotypic data deviated from the expected MMP3, was differentially expressed in preadipocytes/stro- under Hardy-Weinberg equilibrium. The linkage disequilib- mal vascular cells, but the expression of several other rium coefficients (DЈ and r2) between each of the seven MMP genes (MMP7, MMP20, MMP27, MMP8, MMP10, representative variants are given in Table 2. MMP1, MMP12, and MMP13) clustered on 11q22 was not Two representative variants, rs650108 and rs522616, detectable by differential-display PCR. Using differential- were associated with BMI (P ϭ 0.05 and 0.0009, respec- display PCR, we showed that MMP3 mRNA levels were tively, adjusted for age, sex, birth date, family member- decreased in preadipocytes/stromal vascular cells isolated ship, and Pima heritage) when analyzed under a dominant from obese Pima-Indian subjects compared with nonobese model (Table 3). Both of these variants were also associ- subjects, and real-time quantitative PCR showed a signif- ated with type 2 diabetes (P ϭ 0.05 and 0.005, respectively, icant negative correlation between MMP3 mRNA expres- adjusted for age, sex, birth date, family membership, and sion levels and percent body fat. It is possible that Pima heritage) when analyzed under a dominant model technical procedures during the initial adipose tissue (Table 4). Two additional variants, rs3025058 and the biopsy may have resulted in different proportions of Lys45Glu missense mutation (rs679620), were also associ- endothelial “contaminating” cells in the preadipocytes/

TABLE 3 Association analysis of the individual MMP3 variants with BMI in Pima Indians Mean BMI (kg/m2)* P† Variant Alleles MAF 1/1 (n) 1/2 (n) 2/2 (n) Additive Dominant Recessive rs3025058 6T/5T 0.23 36.4 (518) 35.8 (295) 35.3 (33) 0.12 0.24 0.20 rs679620 G/A 0.23 36.2 (506) 35.6 (380) 35.4 (36) 0.20 0.58 0.24 rs3020919 G/A 0.06 35.9 (722) 37.0 (102) 34.6 (2) 0.25 — 0.23 rs650108 T/C 0.28 36.0 (402) 36.3 (314) 33.9 (50) 0.60 0.05 0.70 rs522616 G/A 0.40 36.4 (306) 36.4 (374) 34.4 (126) 0.04 0.0009 0.66 ϩ1494 7T/8T 0.02 36.1 (840) 36.8 (34) — 0.49 — 0.49 Ϫ1475 G/A 0.03 36.3 (812) 34.1 (62) — 0.24 — 0.24 *BMI by variant genotype (1/1, 1/2, or 2/2) is given as age- and sex-adjusted means. †P values were calculated under three models assuming an additive, dominant, or recessive affect of the common allele and were adjusted for age, sex, birth date, family membership, and Pima heritage. MAF, minor allele frequency.

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TABLE 4 Genotype frequencies (nondiabetic versus diabetic) and association analyses with type 2 diabetes for the MMP3 variants in Pimas Nondiabetic Diabetic P* Variant 1/1 (%) 1/2 (%) 2/2 (%) 1/1 (%) 1/2 (%) 2/2 (%) A D R OR (95% CI) rs3025058 194 (65) 88 (29) 19 (6) 324 (60) 207 (38) 14 (3) 0.98 0.005 0.30 2.48 (1.31–4.66) rs679620 181 (63) 88 (31) 18 (6) 325 (60) 199 (37) 17 (3) 0.62 0.02 0.72 2.14 (1.16–3.95) rs3020919 251 (88) 32 (11) 1 (0.35) 471 (87) 70 (13) 1 (0.19) 1.00 0.13 0.98 1.01 (0.59–1.72) rs650108 155 (57) 95 (35) 24 (9) 251 (32) 222 (44) 27 (5) 0.88 0.05 0.26 1.83 (1.01–3.32) rs522616 120 (42) 110 (38) 59 (20) 189 (36) 269 (51) 69 (13) 0.72 0.005 0.18 1.76 (1.19–2.59) ϩ1494 297 (97) 9 (3) 0 (0) 543 (96) 25 (4) 0 (0) 0.32 — 0.32 1.51 (0.67–3.38) Ϫ1475 273 (90) 31 (10) 0 (0) 539 (95) 31 (5) 0 (0) 0.44 — 0.44 1.32 (0.66–2.66) *P values were calculated under three models assuming an additive (A), dominant (D), or recessive (R) effect of the common allele adjusted for age, sex, birth date, and Pima heritage. OR, odds ratio. stromal vascular cells isolated from obese versus nono- representative SNPs that were genotyped for association bese subjects, which could have lead to altered levels of analyses, rs650108 and rs522616 were associated with cell-specific genes. However, we have previously shown both BMI and type 2 diabetes. Because rs650108 is located that the expression level of endothelial cell–specific mol- in intron 8 ϳ500 bases from the splice-donor site and ϳ650 ecule 2, a corollary of endothelial contamination, was bases from the splice-acceptor site, it is unlikely that this consistent among these subjects (14). variant directly contributes to the two phenotypes, but MMPs may play an important role in adipose tissue instead it may be in linkage disequilibrium with a putative remodeling (4,5). In two previous studies, Chavey et al. (4) functional polymorphism(s). The rs522616 polymorphism and Maquoi et al. (5) examined the expression levels of is located in the 5Ј-UTR ϳ709 bases upstream of the various MMPs and TIMPs (tissue inhibitors of MMP) in transcription start site, and it does not appear to be in a adipose tissue from mouse models of obesity. Both studies known conserved regulatory element; however, it is in a found significant changes in MMP and TIMP expression string of 4 bases that are conserved across several species. levels in obese adipose tissue and suggested that these Two additional SNPs, a promoter SNP (rs3025058) and a proteins may be involved in obesity-mediated adipose nonsynonymous coding SNP (rs679620), were not associ- tissue remodeling. MMPs also appear to be important for ated with BMI but were associated with type 2 diabetes. preadipocyte differentiation (adipogenesis) (4,5,15–17). The promoter variant rs3025058 (alternatively termed 5A/ Croissandeau et al. (15) demonstrated that MMP2 and 6A) is a common polymorphism located in a SIRE (strome- MMP9 play a role in the differentiation of murine 3T3-LI lysininterleukin-1–responsiveelement;Ϫ1614G(T)TTTTTCC preadipocytes into mature adipocytes, and Bouloumie et CCCC-ATCAAAGϪ1595) important for interleukin-1–induced al. (16) showed that MMP2 and MMP9 may be involved in DNA binding (19). It has been shown that two proteins modulating murine 3T3F442A adipocyte differentiation. bind to this site, ZBP-89 and nuclear factor-␬B, and that There are a number of recent studies both corroborating binding of ZBP-89 is dependent on the string of Cs (20), and conflicting our finding of decreased expression of whereas the binding of nuclear factor-␬B is dependent on MMP3 in preadipocytes/stromal vascular cells isolated the string of Ts (19). The coding variant rs679620 predicts from obese subjects. For example, Maquoi et al. (18) an amino acid substitution (Lys45 [basic] to Glu [acidic]). demonstrated that MMP3Ϫ/Ϫ mice had increased adipo- Lys45 is part of the 82–amino acid propeptide pro-MMP3, cyte hypertrophy when fed a high-fat diet, and Alexander which is involved in maintaining MMP3 latency (21); et al. (17) showed that MMP3Ϫ/Ϫ (Str1Ϫ/Ϫ) mice had however, the functional importance of this particular accelerated adipocyte differentiation during mammary lysine residue is unclear. These two variants are represen- gland involution. In the same study, Alexander et al. noted tative SNPs for several other polymorphisms that were that MMP3Ϫ/Ϫ mice backcrossed onto another back- detected by sequencing of this gene; however, none of the ground (FVB/N) became obese with age (17). In contrast nongenotyped SNPs appear to be located within any to our study, both Maquoi et al. (5), in an earlier study, and regions that would support a functional role. It should also Chavey et al. (4) found that MMP3 expression levels were be mentioned that the homozygous minor genotype for upregulated in adipose tissue isolated from obese mice. these two variants is somewhat rare (4.87 and 5.22%, The reasons for these disparities with our results are respectively) (data not shown), so it may be possible that unclear. They could possibly be caused by differences in this low frequency is exaggerating the association with model systems (human versus mouse models, or diet- diabetes. induced versus non–diet-induced obesity) or alternatively The promoter polymorphism rs3025058 (5A/6A) has by differences in adipose tissue depots (subcutaneous previously been shown to affect MMP3 promoter activity versus gonadal). in a number of cell lines (22), including human fibroblasts Because MMP3 mapped to a region of linkage to percent (23), rat smooth muscle cells (20), and human macro- body fat and BMI, we sought to determine whether expres- phages (24). In each of these studies, the 5A allele had sion differences were attributable to variation at this locus, higher promoter activity compared with the 6A allele or whether the differential expression was caused by (20,22–24). In contrast, the 5A allele was associated with variation in a protein/factor that regulates MMP3 and the lower MMP3 serum levels, compared with the 6A allele, in position of this gene in a region of linkage was simply human subjects (25). In our study, the promoter variant serendipitous. Sequencing of ϳ13 kb of the MMP3 gene rs522616, but not rs3025058, was associated with BMI; identified 24 sequence variations, many of which were in however, no association was observed between the very high or complete linkage disequilibrium. Among the rs522616 genotype and MMP3 mRNA levels in preadipo-

3164 DIABETES, VOL. 55, NOVEMBER 2006 M.T. TRAURIG AND ASSOCIATES cytes/stromal vascular cells. Therefore, further functional 5. Maquoi E, Munaut C, Colige A, Collen D, Lijnen HR: Modulation of adipose studies are needed to assess the biological relevance of tissue expression of murine matrix and their tissue this SNP. It is possible that this variant has no role in inhibitors with obesity. Diabetes 51:1093–1101, 2002 6. Adeyemo A, Luke A, Cooper R, Wu X, Tayo B, Zhu X, Rotimi C, Bouzekri controlling MMP3 activity, and the position of the MMP3 N, Ward R: A genome-wide scan for body mass index among Nigerian gene within a region of linkage to percent body fat/BMI families. Obes Res 11:266–273, 2003 occurred by chance. In support of this, we found that 7. Platte P, Papanicolaou GJ, Johnston J, Klein CM, Doheny KF, Pugh EW, adjustment of our linkage peak at 11q22 for the effect of Roy-Gagnon MH, Stunkard AJ, Francomano CA, Wilson AF: A study of any of these single SNPs did not reduce the evidence for linkage and association of body mass index in the Old Order Amish. Am J linkage to percent body fat previously observed at this Med Genet 121:71–80, 2003 region (data not shown). Therefore, it is possible the 8. van Tilburg JH, Sandkuijl LA, Strengman E, Pearson PL, van Haeften TW, downregulation of this gene in some obese subjects is Wijmenga C: Variance-component analysis of obesity in type 2 diabetes confirms loci on 1q and 11q. Obes Res 11:1290–1294, 2003 attributable to a change in the level of a protein/hormone 9. Norman RA, Thompson DB, Foroud T, Garvey WT, Bennett PH, Bogardus that affects MMP3 expression, rather than variation in the C, Ravussin E: Genomewide search for genes influencing percent body fat MMP3 gene itself. Alternatively, MMP3 expression may be in Pima Indians: suggestive linkage at chromosome 11q21–q22: Pima too complex (controlled by multiple variants within the Diabetes Gene Group. Am J Hum Genet 60:166–173, 1997 5Ј-UTR of MMP3, as well as being influenced by external 10. Hanson RL, Ehm MG, Pettitt DJ, Prochazka M, Thompson DB, Timberlake factors secondary to obesity, such as insulin), such that we D, Foroud T, Kobes S, Baier L, Burns DK, Almasy L, Blangero J, Garvey were unable to detect an association between a single WT, Bennett PH, Knowler WC: An autosomal genomic scan for loci linked promoter variant and MMP3 expression levels. Therefore, to type II diabetes mellitus and body mass index in Pima Indians. Am J Ϫ/Ϫ Hum Genet 63:1130–1138, 1998 although MMP3 mice become obese, our data in Pima 11. Knowler WC, Bennett PH, Hamman RF, Miller M: Diabetes incidence and Indians cannot distinguish whether downregulation of prevalence in Pima Indians: a 19-fold greater incidence than in Rochester, MMP3 leads to obesity, and/or is a consequence of obesity, Minnesota. Am J Epidemiol 108:497–505, 1978 in humans. In light of our conflicting data that MMP3 12. World Health Organization. Diabetes Mellitus: Report of a WHO Study mRNA levels are negatively correlated with percent body Group. Geneva, World Health Org., 1985 (Tech. Rep. Ser., no. 727) fat in a small number of research subjects, but that a 13. Lee YH, Nair S, Rousseau E, Allison DB, Page GP, Tataranni PA, Bogardus MMP3 promoter variant that is associated with BMI is not C, Permana PA: Microarray profiling of isolated abdominal subcutaneous adipocytes from obese vs non-obese Pima Indians: increased expression of correlated with MMP3 mRNA levels, further studies such inflammation-related genes. Diabetologia 48:1776–1783, 2005 as protein expression and/or activity assays are needed to 14. Permana PA, Nair S, Lee YH, Luczy-Bachman G, Vozarova De Courten B, substantiate our MMP3 expression data. Tataranni PA: Subcutaneous abdominal preadipocyte differentiation in In conclusion, we propose that the MMP3 pathway is vitro inversely correlates with central obesity. Am J Physiol Endocrinol altered in human obesity. We believe this alteration may Metabol 286:E958–E962, 2004 be the result of a combination of genetic variation within 15. Croissandeau G, Chretien M, Mbikay M: Involvement of matrix metallo- the MMP3 locus itself, as well as variation in additional proteinases in the adipose conversion of 3T3–L1 preadipocytes. Biochem J 364:739–746, 2002 factors, either primary or secondary to obesity, that regu- 16. Bouloumie A, Sengenes C, Portolan G, Galitzky J, Lafontan M: Adipocyte late expression of the MMP3 gene. produces matrix metalloproteinases 2 and 9: involvement in adipose differentiation. Diabetes 50:2080–2086, 2001 ACKNOWLEDGMENTS 17. Alexander CM, Selvarajan S, Mudgett J, Werb Z: Stromelysin-1 regulates This research was supported by the Intramural Research adipogenesis during mammary gland involution. J Cell Biol 152:693–703, 2001 Program of the National Institutes of Health, National 18. Maquoi E, Demeulemeester D, Voros G, Collen D, Lijnen HR: Enhanced Institutes of Diabetes and Digestive and Kidney Diseases. nutritionally induced adipose tissue development in mice with stromely- M.T.T. is supported by a Mentor Grant from the American sin-1 gene inactivation. Thromb Haemost 89:696–704, 2003 Diabetes Association. 19. 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