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Cathepsin-D, a Key Protease in Breast Cancer, Is Up-Regulated in Obese Mouse and Human Adipose Tissue, and Controls Adipogenesis

Masson O, Prébois C, Derocq D, Meulle A, Dray C, Daviaud D, Quilliot D, Valet P, Muller C, Liaudet-Coopman E - PLoS ONE (2011)

Bottom Line: Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse.We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers.Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity.

Affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France.

ABSTRACT

The aspartic protease cathepsin-D (cath-D) is overexpressed by human epithelial breast cancer cells and is closely correlated with poor prognosis in breast cancer. The adipocyte is one of the most prominent cell types in the tumor-microenvironment of breast cancer, and clinical studies have shown that obesity increases the incidence of breast cancer. Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse. Cath-D expression is also increased during human and mouse adipocyte differentiation. We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers. Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity.

Cath-D expression increases during 3T3-L1 adipogenesis.Protein expression of cath-D was analysed by immunoblotting in 3T3-L1 adipocytes grown to confluence (D0), and after the differentiation process had been induced for the indicated days. The pro-cath-D secreted over 48h was monitored during differentiation. ERK2 was used as loading control.
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pone-0016452-g003: Cath-D expression increases during 3T3-L1 adipogenesis.Protein expression of cath-D was analysed by immunoblotting in 3T3-L1 adipocytes grown to confluence (D0), and after the differentiation process had been induced for the indicated days. The pro-cath-D secreted over 48h was monitored during differentiation. ERK2 was used as loading control.

Mentions: Obesity is characterized by the increased intracellular accumulation of lipids, a characteristic of adipocyte differentiation that is significantly correlated with adipocyte differentiation. Since cath-D expression was up-regulated in obese tissues, we next investigated whether cath-D plays any role in adipogenesis. We first investigated the regulation of cath-D expression during the course of differentiation of the 3T3-F442A preadipocyte cell line, a valuable model of adipogenesis [38] (Figure 2). Interestingly, cath-D mRNA (Figure 2A) and protein (Figure 2B) expression was progressively up-regulated during adipogenesis. Since the overexpression of cath-D leads to the hypersecretion of the 52 kDa pro-cath-D into the extracellular environment [19], we then investigated whether adipocytes secrete 52 kDa pro-cath-D. Secreted 52 kDa pro-cath-D was only detected in fully-differentiated adipocytes from day 10 of differentiation (Figure 2B). To validate our experimental conditions, we studied in parallel the expression of PPARγ (Figure 2A), HSL (Figure 2B) and aP2 (Figure 2A) adipocyte markers of differentiation. As expected, the levels of these markers increased progressively during the acquisition of the adipocyte phenotype (Figure 2A–B). In addition, the amount of cytoskeletal β-actin protein decreased during adipocyte differentiation, reflecting the change in cellular morphology (Figure 2B), as previously described [39]. Interestingly, a similar up-regulation of cath-D protein expression was observed during 3T3-L1 adipocyte differentiation (Figure 3). It was previously shown that cath-D expression was stimulated by insulin in epithelial breast cancer cells [40], [41]. To ensure that the insulin treatment was not involved in the effect observed, we used parental NIH-3T3 fibroblasts and fully-differentiated 3T3-F442A adipocytes and. As shown in Figure 4, the levels of cath-D protein remained unaffected in the adipogenic differentiation medium both in NIH-3T3 fibroblasts (Figure 4A) and in mature 3T3-F442A adipocytes (Figure 4B). We also confirmed that cath-D protein expression was not altered in 3T3-F442A adipocytes by insulin in dose-response experiments at low serum concentrations (Figure 4C). Altogether, these results strongly suggest that cath-D up-regulation is induced by the differentiation process per se and not by insulin.

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Cathepsin-D, a Key Protease in Breast Cancer, Is Up-Regulated in Obese Mouse and Human Adipose Tissue, and Controls Adipogenesis

Masson O, Prébois C, Derocq D, Meulle A, Dray C, Daviaud D, Quilliot D, Valet P, Muller C, Liaudet-Coopman E - PLoS ONE (2011)

Cath-D expression increases during 3T3-L1 adipogenesis.Protein expression of cath-D was analysed by immunoblotting in 3T3-L1 adipocytes grown to confluence (D0), and after the differentiation process had been induced for the indicated days. The pro-cath-D secreted over 48h was monitored during differentiation. ERK2 was used as loading control.
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pone-0016452-g003: Cath-D expression increases during 3T3-L1 adipogenesis.Protein expression of cath-D was analysed by immunoblotting in 3T3-L1 adipocytes grown to confluence (D0), and after the differentiation process had been induced for the indicated days. The pro-cath-D secreted over 48h was monitored during differentiation. ERK2 was used as loading control.
Mentions: Obesity is characterized by the increased intracellular accumulation of lipids, a characteristic of adipocyte differentiation that is significantly correlated with adipocyte differentiation. Since cath-D expression was up-regulated in obese tissues, we next investigated whether cath-D plays any role in adipogenesis. We first investigated the regulation of cath-D expression during the course of differentiation of the 3T3-F442A preadipocyte cell line, a valuable model of adipogenesis [38] (Figure 2). Interestingly, cath-D mRNA (Figure 2A) and protein (Figure 2B) expression was progressively up-regulated during adipogenesis. Since the overexpression of cath-D leads to the hypersecretion of the 52 kDa pro-cath-D into the extracellular environment [19], we then investigated whether adipocytes secrete 52 kDa pro-cath-D. Secreted 52 kDa pro-cath-D was only detected in fully-differentiated adipocytes from day 10 of differentiation (Figure 2B). To validate our experimental conditions, we studied in parallel the expression of PPARγ (Figure 2A), HSL (Figure 2B) and aP2 (Figure 2A) adipocyte markers of differentiation. As expected, the levels of these markers increased progressively during the acquisition of the adipocyte phenotype (Figure 2A–B). In addition, the amount of cytoskeletal β-actin protein decreased during adipocyte differentiation, reflecting the change in cellular morphology (Figure 2B), as previously described [39]. Interestingly, a similar up-regulation of cath-D protein expression was observed during 3T3-L1 adipocyte differentiation (Figure 3). It was previously shown that cath-D expression was stimulated by insulin in epithelial breast cancer cells [40], [41]. To ensure that the insulin treatment was not involved in the effect observed, we used parental NIH-3T3 fibroblasts and fully-differentiated 3T3-F442A adipocytes and. As shown in Figure 4, the levels of cath-D protein remained unaffected in the adipogenic differentiation medium both in NIH-3T3 fibroblasts (Figure 4A) and in mature 3T3-F442A adipocytes (Figure 4B). We also confirmed that cath-D protein expression was not altered in 3T3-F442A adipocytes by insulin in dose-response experiments at low serum concentrations (Figure 4C). Altogether, these results strongly suggest that cath-D up-regulation is induced by the differentiation process per se and not by insulin.

Bottom Line: Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse.We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers.Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity.

Affiliation: IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, France.

ABSTRACT

Background: The aspartic protease cathepsin-D (cath-D) is overexpressed by human epithelial breast cancer cells and is closely correlated with poor prognosis in breast cancer. The adipocyte is one of the most prominent cell types in the tumor-microenvironment of breast cancer, and clinical studies have shown that obesity increases the incidence of breast cancer. Here, we provide the first evidence that cath-D expression is up-regulated in adipose tissue from obese human beings, as well as in adipocytes from the obese C57BI6/J mouse. Cath-D expression is also increased during human and mouse adipocyte differentiation. We show that cath-D silencing in 3T3-F442A murine preadipocytes leads to lipid-depleted cells after adipogenesis induction, and inhibits of the expression of PPARγ, HSL and aP2 adipocyte differentiation markers. Altogether, our findings demonstrate the key role of cath-D in the control of adipogenesis, and suggest that cath-D may be a novel target in obesity.

View Similar Images In: Results  - Collection
View Article: Medline Plus - Pubmed Central -  PubMed
Show All Figures - Show MeSH
getmorefigures.php?pmc=3032791&rFormat=json&query=null&req=5