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First published online June 8, 2006
doi: 10.1242/10.1242/jcs.02975

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The Wnt antagonist Dickkopf-1 and its receptors are coordinately regulated during early human adipogenesis

C. Christodoulides^*, M. Laudes^*, W. P. Cawthorn, S. Schinner, M. Soos, S. O'Rahilly, J. K. Sethi and A. Vidal-Puig

Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QR, UK

View larger version (28K): [in a new window]   Fig. 1. DKK1 mRNA and Dkk1 protein expression is transiently upregulated during human adipogenesis and correlates with inhibition of canonical Wnt signalling. (A) DKK1 mRNA and protein expression during human adipogenesis. Subcutaneous preadipocytes from eight unrelated subjects were differentiated in vitro and total RNA extracted at the time points indicated. DKK1 mRNA levels were determined by real-time RT-PCR. A plot of the ratio of Dkk1 to p85 protein obtained by densitometry (see Fig. 1B) is also shown alongside the RNA data for comparison. Results are expressed as fold difference relative to baseline (time 0). (B-D) Dkk1 and ß-catenin protein expression during human adipogenesis. Subcutaneous preadipocytes were differentiated in vitro and whole-cell lysates (B), cytosolic (C), or nuclear extracts (D) were obtained at the time points indicated and subjected to SDS-PAGE and western blot analysis. p85 PI 3-kinase and GAPDH were used as loading controls. Results are representative of at least two independent experiments. 0h indicates onset of differentiation (3 days post-confluence); 6h, 12h, 1d, 2d, 4d, 8d respectively indicate 6 hours, 12 hours, 1 day, 2 days, 4 days and 8 days post-induction of differentiation. (E) Expression of Wnt target genes during human adipogenesis. CyclinD1, PPAR and ID2 mRNA levels were determined by real-time RT PCR using RNA from six of the subjects used in A. Rel., relative. **P<0.01, ***P<0.001.

View larger version (43K): [in a new window]   Fig. 2. DKK1 mRNA and protein expression is restricted to the stromal-vascular fraction of human adipose tissue. (A) DKK1 mRNA levels were measured using real-time RT PCR in subcutaneous and omental stromal-vascular cells and mature adipocytes from eight unrelated subjects. (B) Dkk1 protein expression in vivo. Western blot analysis of whole-cell lysates from subcutaneous stromal-vascular cells and mature adipocytes from four unrelated subjects. (C) DKK1 mRNA expression in human and mouse adipose tissues. Total RNA was extracted from stromal-vascular cells and mature adipocytes from four mice (pooled samples), confluent 3T3-L1 cells and one human subject, and RT-PCR for mouse and human DKK1 was performed. RNA isolated from whole mouse embryo and 3T3-L1 cells stably overexpressing human DKK1 were used as positive controls for mouse and human PCRs, respectively. Data were normalised using 18S control. Preads, preadipocytes; Ads, adipocytes; 3T3, 3T3-L1 cells; m, mouse; h, human; +, positive control; **P<0.01.

View larger version (20K): [in a new window]   Fig. 3. Expression of DKK2 and DKK3 during adipogenesis. (A) Expression of Dkk2 and Dkk3 mRNA in mouse adipose tissue was determined using RNA obtained in Fig. 2C. (B) 3T3-L1 preadipocytes were induced to differentiate and total RNA extracted at the time points indicated. Dkk2 and Dkk3 mRNA levels were measured using real-time RT-PCR. Results are expressed as fold difference relative to the baseline (time 0). All results the mean ± s.e.m. of four independent experiments. (C) Human subcutaneous preadipocytes from six unrelated subjects were differentiated in vitro and total RNA extracted at the time points indicated. mRNA levels of DKK2 and DKK3 were determined by real-time RT-PCR. Results are expressed as fold difference relative to baseline (time 0). Preads, preadipocytes; Ads, adipocytes; 3T3, 3T3-L1 cells; m, mouse; h, human; +, positive control. *P<0.05, **P<0.01, ***P<0.001.

View larger version (27K): [in a new window]   Fig. 4. hDkk1 inhibits Wnt signalling and promotes differentiation of 3T3-L1 preadipocytes. (A) Western blot analysis of whole-cell lysates from 3T3-L1 preadipocytes expressing empty vector (EV) or human Dkk1 (hDkk1) and pooled stromal-vascular cells from four mice (mPreads). Human recombinant Dkk1 protein (rDkk1) was used as positive control, and loading efficiency was assessed using p85 PI 3-kinase. (B) Western blot analysis of cytosolic ß-catenin levels during differentiation of control and hDkk1-expressing 3T3-L1 preadipocytes. (C) Effect of hDkk1 on TOPflash reporter activity in 3T3-L1 cells expressing empty vector (EV) or hDkk1. Results are expressed as fold difference relative to EV control. All results are the mean ± s.e.m. of three independent experiments. (D) Oil-Red O staining of EV and hDkk1-expressing 3T3-L1 adipocytes. Cells were induced to differentiate for 8 days using either differentiation medium lacking IBMX (sub-differentiation) or the full differentiation cocktail. (E) Effect of hDkk1 on adipogenic gene expression. Control and hDkk1-expressing 3T3-L1 cells were differentiated sub-maximally using DI and total RNA extracted at the time points indicated. PPAR1, PPAR2 and aP2 mRNA levels were measured using real-time PCR. Results are expressed as fold difference relative to the basal (time 0) value for control. All results are the mean ± s.e.m. of three independent experiments. All comparisons were made against control using Student's t test. PC, pre-confluent; C, confluent; 0, onset of differentiation (2 days post-confluence); 2, 4, 8 indicate 2, 4 and 8 days post-induction of differentiation respectively; RU, relative units; 3T3, 3T3-L1 cells; hDkk1, human Dkk1. *P<0.05, ***P<0.001.

View larger version (14K): [in a new window]   Fig. 5. Expression of Dkk1 receptors in human adipose tissue. (A) LRP6, (B) KRM1 and (C) LRP5 mRNA levels were measured using real-time RT PCR in subcutaneous and omental, stromal-vascular cells and mature adipocytes from 7-8 unrelated subjects. Preads, preadipocytes; Ads, adipocytes. *P<0.05, **P<0.01, ***P<0.001.

View larger version (14K): [in a new window]   Fig. 6. Expression of Dkk1 receptors is regulated during early human adipogenesis. Subcutaneous preadipocytes from eight unrelated subjects were differentiated in vitro and total RNA extracted at the time points indicated (see Table 1). mRNA levels of (A) KRM1 and (B) LRP5 and LRP6 were determined by real-time RT PCR. Results are expressed as fold difference relative to baseline (time 0). *P<0.05, ***P<0.001.

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