Polyamine depletion inhibits apoptosis following blocking of survival pathways in human chondrocytes stimulated by tumor necrosis factor‐α

Abstract

Chondrocyte apoptosis can be an important contributor to cartilage degeneration, thereby making it a potential therapeutic target in articular diseases. To search for new approaches to limit chondrocytic cell death, we investigated the requirement of polyamines for apoptosis favored by tumor necrosis factor-alpha (TNF), using specific polyamine biosynthesis inhibitors in human chondrocytes. The combined treatment of C-28/I2 chondrocytes with TNF and cycloheximide (CHX) resulted in a prompt effector caspase activation and internucleosomal DNA fragmentation. Pre-treatment of chondrocytes with alpha-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, markedly reduced putrescine and spermidine content as well as the caspase-3 activation and DNA fragmentation induced by TNF and CHX. DFMO treatment also inhibited the increase in effector caspase activity provoked by TNF plus MG132, a proteasome inhibitor. DFMO decreased caspase-8 activity and procaspase-8 content, an apical caspase essential for TNF-induced apoptosis. Although DFMO increased the amount of active, phosphorylated Akt, inhibitors of the Akt pathway failed to restore the TNF-induced increase in caspase activity blunted by DFMO. DFMO also reduced the increase in caspase activity induced by staurosporine, but in this case Akt inhibition prevented the DFMO effect. Pre-treatment with CGP 48664, an S-adenosylmethionine decarboxylase (SAMDC) inhibitor markedly reduced spermidine and spermine levels, and provoked effects similar to those caused by DFMO. Finally DFMO was effective even in primary osteoarthritis (OA) chondrocyte cultures. These results suggest that the intracellular depletion of polyamines in chondrocytes can inhibit both the death receptor pathway by reducing the level of procaspase-8, and the apoptotic mitochondrial pathway by activating Akt.

miR-4262 regulates chondrocyte viability, apoptosis, autophagy by targeting SIRT1 and activating PI3K/AKT/mTOR signaling pathway in rats with osteoarthritis

“…It was well-known that TNF-α played a vital role in the pathogenesis of OA, and participated in the progression of cartilage degeneration (29). TNF-α could induce articular chondrocytes death through promoting cell apoptosis and autophagy (30)(31)(32). Several studies also found that cell autophagy had a protective effect on chondrocytes by inhibiting cell death (33,34).…”

The polyamine analogue N1,N11‐diethylnorspermine can induce chondrocyte apoptosis independently of its ability to alter metabolism and levels of natural polyamines

“…SSAT activity was assayed as previously reported (Stanic et al, 2008) and calculated as pmol/ min/mg protein. Caspase-3-like activity was measured by the cleavage of the fluorogenic peptide substrate Ac-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) as previously detailed (Stanic et al, 2006). Since the sequence DEVD represents a substrate for caspase-3 and other effector caspases, this activity has been referred to as caspase-3-like activity.…”

“…Primary cultures of chondrocytes were also used and prepared as described (Stanic et al, 2006). Chondrocytes were cultured in DMEM/F-12 medium and 10% FCS as previously detailed (Stanic et al, 2006). For experiments, growing chondrocytes were incubated in the absence or presence of 10 mM DENSPM, 10 mM spermine, 1 mM DFMO, 1 mM CGP 48664, 500 U/ml TNF, 0.2 mM CHX or other inhibitors as described in the text.…”

“…Primary cultures of chondrocytes were also used and prepared as described (Stanic et al, 2006). Chondrocytes were cultured in DMEM/F-12 medium and 10% FCS as previously detailed (Stanic et al, 2006).…”

“…In particular, survival of chondrocytes, the only cell type in normal mature cartilage, is linked to cartilage integrity, as occurrence of chondrocyte apoptosis has been described and believed to contribute to the pathogenesis of articular diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA; Goggs et al, 2003;Kuhn et al, 2004). Thus, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis by using specific polyamine biosynthesis inhibitors (Stanic et al, 2006) and, more recently, the symmetrically alkylated polyamine analogue N 1 ,N 11 -diethylnorspermine (DENSPM) (Stanic et al, 2008). DENSPM is currently tested as an anticancer agent (Casero and Marton, 2007), but little information is available about the effects of this and other synthetic polyamine analogues in nontumour cells.…”