Wdnm1-like, a new adipokine with a role in MMP-2 activation - PubMed (original) (raw)

Wdnm1-like, a new adipokine with a role in MMP-2 activation

Yu Wu et al. Am J Physiol Endocrinol Metab. 2008 Jul.

Abstract

White adipose tissue functions in energy storage and as an endocrine organ. DNA microarray analysis led us to identify Wdnm1-like, a distant member of the whey acidic protein/four-disulfide core (WAP/4-DSC) family, as a differentiation-dependent gene in white and brown adipogenesis. Wdnm1-like is a novel 6.8-kDa protein, and Western blot analysis reveals secretion into culture media. Wdnm1-like transcript is selectively expressed in adipose tissue and liver and is enriched approximately 500-fold in white adipose depots vs. brown. Cellular fractionation of WAT demonstrates Wdnm1-like transcript expression is restricted to the adipocyte population. Studies in 3T3-L1 preadipocytes, an in vitro model of white adipogenesis, indicate Wdnm1-like transcript increases within 6 h of adipogenic induction with an approximately 17,000-fold increase by day 7. Dramatic upregulation of Wdnm1-like also accompanies white adipogenesis of ScAP-23 preadipocytes and primary preadipocytes. TNF-alpha treatment of 3T3-L1 adipocytes increased Wdnm1-like transcript level 2.4-fold and was attenuated by pretreatment with the p38 MAP kinase inhibitor SB203580. A number of WAP/4-DSC family proteins function as protease inhibitors. This, taken with the role of extracellular remodeling in adipogenesis, led us to address effects of Wdnm1-like on matrix metalloproteinase (MMP) activity. Gelatin zymography of HT1080 fibrosarcoma cells transfected with a Wdnm1-like expression construct revealed markedly increased levels of active MMP-2. Our findings identify a new member of the adipocyte "secretome" that functions to enhance MMP-2 activity. We postulate that Wdnm1-like may play roles in remodeling of the extracellular milieu in adipogenesis, as well as in tumor microenvironments where adipocytes are key stromal components.

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Figures

Fig. 1.

Fig. 1.

Characteristics of Wdnm1-like protein sequence. A: sequence of the 434-bp Wdnm1-like transcript and the encoded 63-amino acid protein (bolded) are shown. The italicized region of the protein sequence corresponds to the region of whey acidic protein/four-disulfide core (WAP/4-DSC) homology. The underlined G in the protein sequence indicates the position of the first intron. B: alignment of the WAP/4-DSC type region of the Wdnm1-like protein with a consensus WAP/4-DSC sequence (ProDom PD026912). C: intron-exon arrangement of the murine Wdnm1-like gene. Open boxes represent the untranslated region, and solid boxes represent the translated region of the transcript. Thin dashed lines indicate introns. D: location of the murine gene for Wdnm1-like and 2 additional WAP/4-DSC type genes on murine chromosome 11. E: alignment of protein sequence for the 3 related WAP/4-DSC type proteins present in a cluster on murine chromosome 11. The canonical cysteines of the WAP/4-DSC motif are shown in bold typeface; those not found in Wdnm1-like are indicated with upward arrows.

Fig. 2.

Fig. 2.

Wdnm1-like encodes a novel 6.7-kDa secreted protein. A: Western blot analysis of in vitro translation products of HA-tagged Wdnm1-like expression construct or empty vector (EV). A sample (10 μl or 2 μl) of a 1:10 dilution of in vitro translation products was subjected to Western blot analysis performed with anti-HA antibody. Positive control (+) is medium from Wdnm1-like-transfected HT1080 cells. Molecular mass markers in kDa are shown at right. B: hydrophobicity analysis of Wdnm1-like protein sequence. Wdnm1-like protein sequence was subjected to Kyte-Doolittle hydrophobicity analysis using DS Gene 2.1 software with a window size setting of 10. The hydrophobicity score is shown on the _y_-axis and the amino acid number on the _x_-axis. C: Western blot analysis of Wdnm1-like protein expression in cells and culture medium. 293T cells were transfected with an HA-tagged Wdnm1-like expression construct or EV, and a portion of the cell lysate or conditioned medium (as described in

materials and methods

) was analyzed by Western blot analysis using an anti-HA antibody (Ab). Coomassie blue gel staining (for medium) or membrane reprobed with anti-cyclophilin A antibody (for cell lysate) are shown as loading controls. Arrows in A and C indicate signal for Wdnm1-like protein. Lanes comprising the single panels shown in A or C were generated from the same Western blot exposure; however, some lanes have been removed and/or rearranged for clarity and/or economy of presentation. Minor adjustments to brightness and/or contrast were utilized for better visualization.

Fig. 3.

Fig. 3.

Adipose tissue is a primary site of Wdnm1-like expression in vivo. A: real-time PCR assessment of Wdnm1-like transcript level in a panel of murine tissues. Transcript level in muscle tissue was set to a value of 1. *P < 0.001 for white adipose tissue (WAT) or liver vs. the other tissue samples. Sal. gland, salivary gland. B: expression of Wdnm1-like and selected adipocyte marker transcripts in adipose tissue depots. Brown adipose tissue (BAT) (B), subcutaneous (SC) (S), epididymal (E) and retroperitoneal (R) WAT cDNA were used for real-time PCR analysis for transcript level of Wdnm1-like (left), for resistin (Retn, middle) or stearoyl-CoA desaturase 1 (SCD1) and adipose fatty acid-binding protein (aFABP, right). The transcript level in BAT was set to a value of 1. *P < 0.001 for Wdnm1-like transcript level in S, E, and R compared with the B depot. C: cellular fractionation of WAT. cDNA derived from the cells of the stromal vascular fraction (SVF) or adipocyte fraction (AF) of SC WAT was used for real-time PCR analysis of Wdnm1-like (left). Transcript levels of SCD1 (middle) and collagen 1A1 (Col1A1, right) are shown to validate effective fractionation of WAT. The transcript level in SVF was set as a value of 1 for the left and middle panels and for AF for the right panel. *P < 0.001 for AF vs. SVF. D: real-time PCR analysis of Wdnm1-like transcript in wild-type C57BL/6 mice (WT) and ob/ob mice. The value in the respective WT depot was set to 1, *P < 0.002 for ob/ob vs. wild-type. S, E, and B are as defined in B.

Fig. 4.

Fig. 4.

Wdnm1-like transcript increases early and is sustained throughout 3T3-L1 adipocyte differentiation. A: daily time course of transcript expression. RNA was harvested from postconfluent 3T3-L1 preadipocytes before induction of adipogenesis (0) and at indicated daily time points after dexamethasone (Dex)/methylisobutylxanthine (MIX) induction of adipogenesis. A sample (5 μg) of total RNA was analyzed by Northern blot using Wdnm1-like, SCD1, aFABP, and peroxisome proliferator-activated receptor-γ (PPARγ) radiolabeled cDNA probes. For Wdnm1-like signal, shorter (S) and longer (L) autoradiographic exposure times are shown. EtBr staining of rRNA is shown as a gel loading control. Minor adjustments to brightness and/or contrast were utilized for better visualization. B: real-time PCR analysis of Wdnm1-like transcript levels in 3T3-L1 (left) or ScAP-23 (right) preadipocytes (P) and adipocytes (A). Value in the respective preadipocyte sample was set to 1, and *P < 0.001 for A vs. P. The signals for the 3T3-L1 and ScAP-23 preadipocyte samples were undetectable after 40 PCR cycles and were set to a value of 40, as described in

materials and methods

, such that fold changes could be calculated. C: upregulation of Wdnm1-like transcript (left) in primary murine preadipocytes (P) or following differentiation to adipocytes (A). Levels of the adipocyte markers SCD1 and aFABP (right) in either preadipocytes (P) or adipocytes (A), confirming effective adipogenesis of primary cultures. Transcript levels in preadipocytes were set to value of 1, *P < 0.001 for preadipocytes vs. adipocytes. D: transcript analysis in early adipogenesis. Real-time PCR for the indicated transcript was conducted on samples harvested at 24 h before adipogenic induction (−24), at the time of adipogenic induction (0), or postadipogenic induction (6, 12, 24, 36, and 48 h). For each transcript, the level in the respective time 0 sample was set to 1. For those samples whose values are not evident from the graph, the numbers directly above the respective _x_-axis indicate fold induction.

Fig. 5.

Fig. 5.

Regulation Wdnm1-like transcript by components of the adipogenic cocktail, TNF-α and LPS. A: regulation of Wdnm1-like transcript expression by components of the adipogenic cocktail. Postconfluent 3T3-L1 cells (0) were treated with 1 μM Dex (D) or 0.5 mM MIX (M) or 1 μM Dex and 0.5 mM MIX (DM) in combination for 1 day (day 1), 2 days (day 2), or for 2 days followed by culture for 3 days (day 5). In each case, RNA was harvested 5 days postinduction. Northern blot analysis was performed on 5 μg total RNA using a radiolabeled Wdnm1-like probe. For Wdnm1-like signal, shorter (S) and longer (L) autoradiographic exposures are shown. EtBr staining of rRNA is shown as a gel loading control. Minor adjustments to brightness and/or contrast were utilized for better visualization. B: TNF-α upregulates Wdnm1-like transcript in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pretreated with either DMSO vehicle (V) or the indicated pharmacological inhibitors of intracellular signaling pathways, and, after 1 h, TNF-α was added to cultures at 10 ng/ml, as described in

materials and methods

. After 16 h, RNA was analyzed for Wdnm1-like transcript level by real-time PCR. Value in vehicle-treated adipocytes was set to 1, *P < 0.001 for TNF-α vs. vehicle and for TNF-α vs. SB + TNF-α; #_P_ > 0.05 for WM + TNF-α vs. TNF-α alone and for RAP + TNF-α vs. TNF-α alone; **P < 0.001 for SB vs. TNF-α; ##_P_ > 0.05 for WM and RAP vs. vehicle. SB, SB203580; WM, wortmannin; RAP, rapamycin. C: LPS upregulates Wdnm1-like transcript in RAW264.7 murine macrophages. RNA was harvested from control cells (−) or those treated for 4 h with LPS (+), with duplicate samples shown. Transcript level for Wdhm1-like was undetectable in the (−) samples, and, as described in

materials and methods

, a value of 40 cycles was assigned to calculate fold differences. *P < 0.001 for (+) vs. (−) samples.

Fig. 6.

Fig. 6.

Secreted Wdnm1-like increases levels of active matrix metalloproteinase (MMP)-2. A: left: ectopic expression of Wdnm1-like transcript in HT1080 cells by real-time PCR. Right: ectopic expression of Wdnm1-like protein in HT1080 cells by Western blot analysis. Cell lysate (for RNA) or medium (for protein) was harvested at 48 h posttransfection of HT1080 cells with either empty vector (EV) control or the Wdnm1-like-HA expression construct and subjected to Western blot analysis using anti-HA antibody. Coomassie blue gel staining is shown as a loading control. B: gelatin zymography analysis of media from HT1080 cells transfected with Wdnm1-like-HA expression construct or EV control. Medium was collected 48 h posttransfection and analyzed on 0.1% gelatin zymograph gels as described in

materials and methods

. Representative result of a minimum of 3 independent experiments is shown. Location of the pro-, intermediate (int), and active MMP-2 are indicated at right. C: real-time PCR analysis of transcript levels of MMP-2 (left) and MT1-MMP/MMP-14 (right) in cells transfected with an EV or the Wdnm1-like-HA expression construct. A_–_C: duplicate samples derived from independent transfections are shown. A and C: the left EV sample was set to 1. Lanes comprising panels shown for either A (right) or for B were generated from the same Western blot exposure (B) or zymograph gel (C); however, some lanes have been removed and/or rearranged for clarity and/or economy of presentation. Minor adjustments to brightness and/or contrast were utilized for better visualization.

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