Protease Inhibitor Treatments Reveal Specific Involvement of Matrix Metalloproteinase-9 in Human Adipocyte Differentiation

Abstract

We previously showed that human and murine 3T3-F442A preadipocytes produced and released matrix metalloproteinases (MMPs) 2 and 9 and that a treatment by MMP inhibitors resulted in the blockade of murine fat cell adipose conversion. In parallel, investigators reported that other protease inhibitors, the human immunodeficiency virus (HIV) protease inhibitors (PIs) involved in lipodystrophy in humans, also reduced the adipocyte differentiation process of several murine cell lines. The present work was performed to define the effects of MMP inhibitors and HIV-PIs on the human adipocyte differentiation process, to clarify the involvement of MMPs in the control of human adipogenesis, and to determine whether HIV-PIs interact with MMPs in the control of this process. The effect of two MMP inhibitor and four HIV-PI treatments on the differentiation of primary culture human preadipocytes, as well as the putative relationships between HIV-PIs and MMP-2 and -9 expression, release, or activity were investigated. We showed that MMP inhibitors and HIV-PIs reduced the human adipocyte differentiation process as assessed by the decrease of cell protein and/or triglyceride contents and expression of fatty acid binding protein and hormone-sensitive lipase, two adipocyte markers. Unlike MMP inhibitors, HIV-PIs were devoid of any effect per se on recombinant MMP-2 and 9 activities but reduced the expression and release of MMP-9 by human preadipocytes. Thus, the present study indicates that the modulation of the extracellular matrix components through the production and/or activity of MMPs, and, more precisely, MMP-9 might be a key factor in the regulation of human adipose tissue development.The mechanisms responsible for the growth of adipose tissue (AT) are still not well defined. Adipocyte hypertrophy and hyperplasia, due to the recruitment and differentiation of adipocyte precursor cells, or preadipocytes, into adipocytes are major cellular events in the development of the fat mass. Together with the hypertrophy and the hyperplasia events, recent investigations have clearly demonstrated that angiogenesis and extracellular matrix (ECM) remodeling are required for coordinated growth of the fat depot (Lijnen et al., 2002;Rupnick et al., 2002). Among the enzymes involved in the degradation of the ECM components, the matrix metalloproteinase (MMP) family is considered to play a major role (Sternlicht and Werb, 2001). In a previous report, we demonstrated that mature human adipocytes and preadipocytes produce and release two members of the MMP family, MMP-2 and -9, the expression and activity of which were shown to be dependent on the adipocyte differentiation process (Bouloumié et al., 2001). Interestingly, we also reported that treatment of the murine 3T3-F442A preadipocytes, which also produced MMP-2 and -9 during adipose conversion, with MMP inhibitors decreased the rate of adipocyte differentiation, suggesting that MMP activities are required for adipogenesis in rodents. However, no data are available concerning the poten...

Inhibition of Human Preadipocyte Proteasomal Activity by HIV Protease Inhibitors or Specific Inhibitor Lactacystin Leads to a Defect in Adipogenesis, Which Involves Matrix Metalloproteinase-9

“…We previously reported, in accordance with in vivo studies performed on adipose tissue biopsies from HIV-infected lipodystrophic patients (Bastard et al, 2002;Lloreta et al, 2002), that PIs reduced the differentiation of human preadipocytes (i.e., adipocyte precursors) in primary culture isolated from subcutaneous abdominal adipose tissue (AT) (Bourlier et al, 2005). This effect was attributed to an indirect action of HIV-PIs on the expression and secretion of the matrix metalloproteinase-9 (MMP-9), an enzyme implicated in extracellular matrix remodeling and significantly involved in the human adipogenic process in vitro (Bourlier et al, 2005).…”

“…MMP-9 activity has been shown 1) to be involved in the human adipocyte differentiation process (Bouloumié et al, 2001;Bourlier et al, 2005) and 2) to be reduced in human preadipocytes treated by HIV-PIs (Bourlier et al, 2005). To evaluate whether the inhibition of the proteasome per se could affect MMP-9 secretion and expression, overnight-conditioned medium from preadipocytes treated with 0.5 M lactacystin for 10 days was collected to be analyzed by gelatin zymography, and real-time RT-PCR analysis was performed on mRNAs.…”

“…The degradation of these two well-known IB family members leads to NF-B translocation to the nucleus (Whiteside and Israël, 1997) and transcription of NF-B target genes, among them MMP-9 (StPierre et al, 2004). Human preadipocytes were placed in an adipogenic medium and treated for 10 days with lactacystin or various HIV-PIs at concentrations for which no cytotoxic effect was detected and the inhibition of the differentiation was maximal according to our previous publication (Bourlier et al, 2005). Cellular extracts were obtained and analyzed by the Western blot technique.…”

“…This effect was attributed to an indirect action of HIV-PIs on the expression and secretion of the matrix metalloproteinase-9 (MMP-9), an enzyme implicated in extracellular matrix remodeling and significantly involved in the human adipogenic process in vitro (Bourlier et al, 2005). Because the MMP-9 promoter contains an NF-B site (Sato and Seiki, 1993;St-Pierre et al, 2004) and because proteasomal activity is involved in the activation of NF-B pathway, we investigated the potential role of the proteasome in the antiadipogenic effect of HIV-PIs.…”

Macrophages Transmit Human Immunodeficiency Virus Type 1 Products to CD4-Negative Cells: Involvement of Matrix Metalloproteinase 9

“…6B). Besides blocking virus maturation by inhibiting Gag p55 processing, PIs induce a number of cellular effects, including inhibition of proteasome functions (3,29) and metalloproteinase activity (6,51). To establish whether MMPs were indeed involved in the inhibition of transmission of HIV-1 products, we treated the cocultures for 6 h with different concentrations of two broad spectrum inhibitors of MMPs, i.e., GM6001 and MMP IV.…”