Differential Expression and Function of Stamp Family Proteins in Adipocyte Differentiation

Six transmembrane protein of prostate (Stamp) proteins play an important role in prostate cancer cell growth. Recently, we found that Stamp2 has a critical role in the integration of inflammatory and metabolic signals in adipose tissue where it is highly expressed and regulated by nutritional and metabolic cues. In this study, we show that all Stamp family members are differentially regulated during adipogenesis: whereas Stamp1 expression is significantly decreased upon differentiation, Stamp2 expression is increased. In contrast, Stamp3 expression is modestly changed in adipocytes compared to preadipocytes, and has a biphasic expression pattern during the course of differentiation. Suppression of Stamp1 or Stamp2 expression both led to inhibition of 3T3-L1 differentiation in concert with diminished expression of the key regulators of adipogenesis - CCAAT/enhancer binding protein alpha (C/ebpa) and peroxisome proliferator-activated receptor gamma (Ppar?). Upon Stamp1 knockdown, mitotic clonal expansion was also inhibited. In contrast, Stamp2 knockdown did not affect mitotic clonal expansion, but resulted in a marked decrease in superoxide production that is known to affect adipogenesis. These results suggest that Stamp1 and Stamp2 play critical roles in adipogenesis, but through different mechanisms. This is an open-access article distributed under the terms of the Creative Commons Attribution License

The role of Mitogen Activated Protein Kinase (MAPK) Phosphatases (MKPs) in adipogenesis : A study of Vaccinia H1-related (VHR) phosphatase

Animals store most excess energy as fat in specialized cells called adipocytes which form the major part of adipose tissue. Since obesity, which has reached epidemic proportions in the last decades, is the result of excess growth of the adipose tissue caused by both hypertrophy and hyperplasia, understanding how adipocytes expand is an important area research. Adipogenesis, the development of progenitor cells to mature adipocytes, has been widely studied with mouse models and in particular with differentiation of the murine preadipocytes 3T3-L1 and 3T3-F22A. Previous research has found Mitogen Activated Protein kinase (MAPK) activity to be required in the early stages of adipogenesis. MKPs are phosphatases with MAPKs as their substrate. They are known to dephosphorylate MAPKs in several tissues. MKP1 has been shown to have a role in adipogenesis as it can regulate phosphorylation of the MAPK ERK in differentiating 3T3-L1 cells. Previous findings in our group have shown many MKPs to be regulated at the mRNA level during 3T3-L1 differentiation; one of these, MKP5, has been shown to enhance adipocyte differentiation when ectopically expressed. In this study we found that one of the other MKPs, Vaccinia H1-related (VHR) phosphatase is regulated during adipogenesis. We also validate the integrity of two kinds of expression vector constructs created for three MKPs (VHR, MKP6 and VH5) all of which have been shown to be regulated during adipogenesis. The vector constructs for VHR were used in characterization of its potential role in differentiation of 3T3-L1 cells. These results further underscore the importance of MKPs in adipogenesis and pave the way for future studies

<i>Stamp1</i> or <i>Stamp2</i> knockdown reduces 3T3-L1 adipogenesis.

<p>(A–B) Oil Red O staining of WT, sh-GFP, sh-St1 and sh-St2 cells differentiated with pioglitazone (Pio) or vehicle (Ctrl). (A) Representative images of the staining. (B) Quantification from three independent experiments, n = 9. *p<0.05 compared to sh-GFP cells; #p<0.05 between Ctrl and Pio groups. (C) Relative cell number of the same cells as in (A). The results are from three independent experiments, n = 9. *p<0.05 compared to sh-GFP cells; #p<0.05 between Ctrl and Pio groups. (D) Relative cell number of sh-St1 cells differentiated with increasing concentrations of Pio. The results are from one experiment, n = 3. *p<0.05 compared to shGFP; #p<0.05 between brackets.</p

Regulation of Stamp family expression during 3T3-L1 adipogenesis.

<p>(A–C) qRT-PCR analysis of 3T3-L1 cells harvested at the indicated time points (days) of differentiation. The figures show the mRNA expression of <i>Stamp1</i> (A), <i>Stamp2</i> (B, top), and <i>Stamp3</i> (C) normalized to the reference gene <i>36B4</i>. The results are from three independent experiments, n = 9. *p<0.05 compared to d0; #p<0.05 between brackets. (B, bottom) Western analysis showing Stamp2 and β-actin protein levels from day 0 to day 8 of differentiation in 3T3-L1 cells harvested in parallel to those used for qRT-PCR analysis. The figure presented is representative of two independent experiments.</p

<i>Stamp1</i> or <i>Stamp2</i> knockdown reduces <i>Pparγ</i> and <i>aP2</i>, but not <i>C/ebpα</i>, mRNA expression in 3T3-L1 adipocytes.

<p>(A–F) qRT-PCR analysis of WT, sh-GFP, sh-St1 and sh-St2 cells harvested at day 0 (d0) and day 8 of differentiation with (d8+Pio) or without (d8) Pio. The figures show the relative mRNA expression of <i>Stamp1</i> (A), <i>Stamp2</i> (B), <i>Stamp3</i> (C), <i>aP2</i> (D), <i>Pparγ</i> (E), and <i>C/ebpα</i> (F) normalized to the reference gene <i>36B4</i> from three independent experiments, n = 9. *p<0.05 compared to sh-GFP cells; #p<0.05 between brackets.</p

<i>Stamp1</i> or <i>Stamp2</i> knockdown in 3T3-L1 cells.

<p>(A–B) qRT-PCR analysis of WT, sh-GFP, sh-St1 and sh-St2 cells. The figures show relative mRNA expression of <i>Stamp1</i> (A) and <i>Stamp2</i> (B) normalized to the reference gene <i>36B4</i> from one experiment, n = 3. *p<0.05 compared to sh-GFP cells. The data presented are representative of three independent experiments. (C) Western analysis showing Stamp2 and β-actin protein levels at day 8 of differentiation in WT, sh-GFP and sh-St2 cells. The data presented are representative of two independent experiments.</p

<i>Stamp1</i> or <i>Stamp2</i> knockdown does not affect C/ebpβ protein expression in early 3T3-L1 adipogenesis.

<p>(A) Western analysis showing C/ebpβ protein levels at day 0 (d0), day 1 (d1) and day 2 (d2) of differentiation with the same cells as in (5A). (B) Quantification of Western analysis results in (A) with relative C/ebpβ protein levels normalized to β-actin from two independent experiments, n = 6. #p<0.05 between brackets.</p

<i>Stamp1</i> or <i>Stamp2</i> knockdown reduces Pparγ and C/ebpα protein expression in 3T3-L1 adipocytes.

<p>(A) Western analysis showing Pparγ, C/ebpα and β-actin protein levels at day 0 (d0), day 4 (d4) and day 8 (d8) of differentiation in WT, sh-GFP, sh-St1 and sh-St2 cells, plus cells at day 8 differentiated with Pio (d8+ Pio). The data presented are representative of two independent experiments. (B–C) Quantification of Westerns in (A) with relative Pparγ (B) and C/ebpα (C) protein levels normalized to β-actin from two independent experiments, n = 6. *p<0.05.</p

<i>Stamp2</i> knockdown reduces superoxide production in 3T3-L1 cells independent of adipocyte differentiation.

<p>(A–B) NBT assay with WT, sh-GFP, sh-St1 and sh-St2 cells differentiated with (Pio) or vehicle (Ctrl). (A) Representative images of the staining. (B) Quantification from three independent experiments, n = 9. *p<0.05 compared to sh-GFP cells; #p<0.05 between Ctrl and Pio groups.</p

Inflammation and ER stress differentially regulate STAMP2 expression and localization in adipocytes

Background Chronic ER stress and dysfunction is a hallmark of obesity and a critical contributor to metaflammation, abnormal hormone action and altered substrate metabolism in metabolic tissues, such as liver and adipocytes. Lack of STAMP2 in lean mice induces inflammation and insulin resistance on a regular diet, and it is dysregulated in the adipose tissue of obese mice and humans. We hypothesized that the regulation of STAMP2 is disrupted by ER stress. Methods 3T3-L1 and MEF adipocytes were treated with ER stress inducers thapsigargin and tunicamycin, and inflammation inducer TNFα. The treatments effect on STAMP2 expression and enzymatic function was assessed. In addition, 3T3-L1 adipocytes and HEK cells were utilized for Stamp2 promoter activity investigation performed with luciferase and ChIP assays. Results ER stress significantly reduced both STAMP2 mRNA and protein expression in cultured adipocytes whereas TNFα had the opposite effect. Concomitant with loss of STAMP2 expression during ER stress, intracellular localization of STAMP2 was altered and total iron reductase activity was reduced. Stamp2 promoter analysis by reporter assays and chromatin immunoprecipitation, showed that induction of ER stress disrupts C/EBPα-mediated STAMP2 expression. Conclusion These data suggest a clear link between ER stress and quantitative and functional STAMP2-deficiency