Acid beta-glucosidase 1 counteracts p38delta-dependent induction of interleukin-6: possible role for ceramide as an anti-inflammatory lipid

Abstract

Activation of protein kinase C (PKC) by the phorbol ester (phorbol 12-myristate 13-acetate) induces ceramide formation through the salvage pathway involving, in part, acid beta-glucosidase 1 (GBA1), which cleaves glucosylceramide to ceramide. Here, we examine the role of the GBA1-ceramide pathway, in regulating a pro-inflammatory pathway initiated by PKC and leading to activation of p38 and induction of interleukin 6 (IL-6). Inhibition of ceramide formation by fumonisin B1 or down-regulation of PKCdelta potentiated PMA-induced activation of p38 in human breast cancer MCF-7 cells. Similarly, knockdown of GBA1 by small interfering RNAs or pharmacological inhibition of GBA1 promoted further activation of p38 after PMA treatment, implicating the GBA1-ceramide pathway in the termination of p38 activation. Knockdown of GBA1 also evoked the hyperproduction of IL-6 in response to 4beta phorbol 12-myristate 13-acetate. On the other hand, increasing cellular ceramide with cell-permeable ceramide treatment resulted in attenuation of the IL-6 response. Importantly, silencing the delta isoform of the p38 family significantly attenuated the hyperproduction of IL-6. Reciprocally, p38delta overexpression induced IL-6 biosynthesis. Thus, the GBA1-ceramide pathway is suggested to play an important role in terminating p38delta activation responsible for IL-6 biosynthesis. Furthermore, the p38delta isoform was identified as a novel and predominant target of ceramide signaling as well as a regulator of IL-6 biosynthesis.

FIGURE 1.

Differential roles of PKCs in p38 modulation in MCF-7 cells. A, MCF-7 cells were transfected with 5 nm siRNAs of SCR or PKCδ for 48 h and then stimulated with 100 nm PMA for 0.5 h. B, MCF-7 cells were treated with the indicated concentration of Gö6976 (Gö) for 30 min and then stimulated with 100 nm PMA for 0.5 h. Whole cell lysates were prepared and subjected to immunoblot analysis with antibodies for phospho-p38. Equal amounts of protein were loaded in each lane.

FIGURE 2.

Effects of ceramide on p38 phosphorylation induced by PMA or TNF-α in A549 cells. A549 cells were treated with 20 μm C₆-ceramide for 2 h and then stimulated with 100 nm PMA or 25 ng/ml TNF-α for 30 min. Whole cell lysates were prepared and subjected to immunoblot analysis with antibodies for phospho-p38. Equal amounts of protein were loaded in each lane.

FIGURE 3.

Effects of knockdown of GBA1 and of conduritol B epoxide on p38 activation. MCF-7 cells were transfected with 5 nm siRNAs of each of three individual sequences separately for 48 h and then stimulated with 100 nm PMA for 1 h (A) or the indicated periods (B). MCF-7 cells were pretreated with 100 or 200 μm conduritol B epoxide (CBE) for 2 h and then stimulated with 100 nm PMA for 1 h (C). Whole cell lysates were prepared and subjected to immunoblot analysis with antibodies specific for p38α (A and C), phospho-p38 (A–C), and phospho-ERK (B). Equal amounts of protein were loaded in each lane.

FIGURE 4.

Role for ceramide in PMA-induced generation of IL-6. MCF-7 cells were stimulated with 100 nm PMA for the indicated periods, and then levels of IL-6 in culture supernatants were measured using the ELISA system (A). MCF-7 cells were transfected with the indicated siRNAs (SCR, open column; GBA1-a, gray-filled column; GBA1-b, filled column) for 48 h and then stimulated with 100 nm PMA for 3 (C) or 12 h (B). B, levels of IL-6 in culture supernatants were measured using the ELISA system. The data represent mean ± S.E. of 10 values. C, cellular RNAs were extracted and subjected to the Q-RT-PCR as described under “Experimental Procedures.” Levels of IL-6 mRNA were expressed as arbitrary units (AU). The data represent means of two values, and each bar is given with the range. D, MCF-7 cells were transfected with 5 nm siRNAs of the indicated siRNAs (SCR, open column; GBA2, gray-filled column; GBA1-b, filled column) for 48 h and then stimulated with 100 nm PMA for 12 h. Levels of IL-6 in culture supernatants were measured using ELISA. E, MCF-7 transfected with 5 nm siRNAs (SCR, open circle; GBA1-b, closed circle) were stimulated with 100 nm PMA for 12 h following treatment with vehicles (0.1% ethanol) or the indicated concentrations of C₆-ceramide for 30 min. Levels of IL-6 in culture supernatants were measured using an ELISA. The data represent mean ± S.E. of two to three values.

FIGURE 5.

Effects of knockdown of GBA1 by siRNAs on TNF-α or PMA-induced generation of IL-6 in A549 cells. A549 cells were stimulated with 25 ng/ml TNF-α or 100 nm PMA for the indicated periods (A). A549 cells transfected with 5 nm siRNAs of SCR or GBA1-a for 48 h and then stimulated with 25 ng/ml TNF-α or 100 nm PMA for 14 h (B). Levels of IL-6 in culture supernatants were measured using an ELISA. The data represent mean ± S.E. of four values.

FIGURE 6.

Involvement of p38δ in the production of IL-6 in GBA1-silenced cells. A, MCF-7 cells were transfected with 5 nm SCR (open column) or GBA1 (gray-filled column) siRNAs for 48 h following pre-treatment with the indicated concentration of SB202190 for 30 min prior to stimulating cells with or without 100 nm PMA for 12 h. Levels of IL-6 in culture supernatants were measured using an ELISA. The data represent mean ± S.E. of six values. B, MCF-7 cells were transfected with 5 nm SCR, GBA1-b, p38δ-a, or GBA1-b and p38δ-a for 48 h, and then the effectiveness of siRNA treatment was assessed by immunoblot analysis with antibodies specific for p38δ or GBA1. The transfected cells were further stimulated with 100 nm PMA for 12 (C) and 1.5 h (D). Levels of IL-6 in culture supernatants (C), and mRNA levels of IL-6 (gray-filled column) and p38δ (open column) (D) were measured. The data regarding IL-6 (C) or IL-6 mRNA (D) represent mean ± S.E. of six values or mean of two values ± the range, respectively. E, inhibition of IL-6 generation by 10 μm SB202190 or p38δ siRNA in PMA stimulation of GBA1-silenced cells is expressed as the percentage relative to dimethyl sulfoxide or SCR treatment in PMA stimulation of GBA1-silenced cells, respectively. The values are calculated from the data shown in A and C. The data represent mean ± S.E. of at least four values.

FIGURE 7.

Regulation of p38δ in GBA1-knocked down cells. A, MCF-7 cells were transfected with 5 nm SCR, GBA1-b, or GBA1-b and p38δ-a for 48 h, and then stimulated with 100 nm PMA for 30 min. Equal amounts of protein for phospho-p38 immunoblot analysis were loaded in each lane. B, recombinant phosphorylated/active p38δ proteins were incubated with recombinant human PP1c-γ isoform or PP2Ac. p38δ phosphorylation was detected by Western blotting using phospho-p38 antibodies.

FIGURE 8.

Effects of p38δ overexpression on IL-6 biosynthesis in MCF-7 cells. MCF-7 cells were transfected with the indicated amounts of human p38δ-FLAG pcDNA3.0 vectors or 300 ng of DNA of empty vectors shown as “0” of human p38δ-FLAG pcDNA3.0 vectors (300 ng) followed by treatment with or without 100 nm PMA for 12 h. Levels of IL-6 in culture supernatants were measured using an ELISA. The data represent mean ± S.E. of three values.

FIGURE 9.

Negative regulation of IL-6 induction by the salvage pathway of ceramide formation. Red or blue lines indicate the pathway of negative or positive regulation for IL-6 induction, respectively. ACD, acid ceramidase; ASM, acid sphingomyelinase; cPKC, classical PKC; CS, ceramide synthase.