To analyze proteolytic activation of CREB3L1, we fractionated human hepatoma Huh7 cells (Nakabayashi et al., 1982) into membrane and nuclear fractions, and used an antibody reacting against the NH 2 -terminal domain of CREB3L1 (Denard et al., 2011) to examine the cleavage of CREB3L1 through immunoblot analysis. In the absence of doxorubicin, CREB3L1 existed as the full length precursor (∼80 kDa) in membranes and the cleaved nuclear form of CREB3L1 (∼55 kDa) was barely detectable (Figure 1B, lane 1). Treatment with doxorubicin markedly raised the amount of the nuclear form of CREB3L1 (Figure 1B, lane 2). The amount of membrane protein calnexin and nuclear protein lysine-specific demethylase 1 (LSD1) was not altered by doxorubicin treatment (Figure 1B). Doxorubicin may increase the amount of nuclear CREB3L1 through stimulation of CREB3L1 precursor cleavage or inhibition of nuclear CREB3L1 degradation, which was reported to be carried out by proteasomes (Murakami et al., 2009). To determine whether doxorubicin inhibits degradation of nuclear CREB3L1, we transfected Huh7 cells with a cDNA encoding NH 2 -terminal fragment of CREB3L1 resembling the cleaved nuclear form of the protein (pCMV-CREB3L1(Δ381-519)) (Denard et al., 2011). The amount of transfected nuclear form of CREB3L1 was not affected by doxorubicin (Figure 1C, lanes 2 and 3). However, this amount was increased in cells treated with the proteasome inhibitor MG132 (Figure 1C, lanes 5 and 6), suggesting that overexpression of the transfected protein did not overwhelm the machinery that degrades nuclear CREB3L1. These results suggest that doxorubicin does not stabilize nuclear CREB3L1. Thus, doxorubicin appears to increase nuclear CREB3L1 by stimulating proteolysis of its precursor.

To determine whether doxorubicin-stimulated cleavage of CREB3L1 was catalyzed by S1P and S2P, we analyzed the cleavage in mutant Chinese Hamster Ovary (CHO) cells deficient in S1P or S2P (Rawson et al., 1997, 1998). In wild type CHO cells, doxorubicin stimulated cleavage of CREB3L1 to produce the nuclear form (Figure 1D, lane 2). In contrast, doxorubicin failed to produce the nuclear form of CREB3L1 in mutant cells deficient in either S1P or S2P (Figure 1D, lanes 4 and 6). In wild type CHO cells, we also detected in the membrane fraction a cleaved fragment with a molecular weight similar to that of the nuclear form (Figure 1D, lanes 1 and 2). This fragment was absent in cells deficient in S1P (Figure 1D, lanes 3 and 4) but dramatically elevated in cells deficient in S2P (Figure 1D, lanes 5 and 6). These findings suggest that this membrane-bound fragment is the intermediate form of CREB3L1 that was cleaved by S1P but not by S2P. Similar cleavage intermediates were observed in earlier studies of SREBP-2, a prototypes of RIP substrates, in mutant CHO cells deficient in S2P (Rawson et al., 1997; Ye et al., 2000). SREBP-2 was cleaved in sterol-depleted CHO cells (Figure 1E, lane 1) to activate genes required for cholesterol synthesis and uptake (Brown and Goldstein, 2009). However, this cleavage was not activated by doxorubicin (Figure 1E, lanes 4 and 5). Thus, doxorubicin appears to specifically induce proteolytic activation of CREB3L1.

An alternative approach to determine the effect of doxorubicin on proteolytic activation of CREB3L1 is to analyze the effect of the compound on expression of target genes activated by CREB3L1. In Huh7 cells transfected with a control shRNA (Huh7-shControl), doxorubicin induced the expression of collagen 1α1 and p21 (Figure 2A,B), both of which were shown to be direct targets of CREB3L1 (Murakami et al., 2009; Denard et al., 2011). In Huh7 cells stably transfected with a shRNA targeting CREB3L1 (Huh7-shCREB3L1) (Denard et al., 2011) in which expression of CREB3L1 was drastically reduced (Figure 2C), induction of these genes was markedly blunted (Figure 2A,B).

Figure 2 Download asset Open asset Doxorubicin induces transcription of genes activated by CREB3L1. (A),(B) On day 0, indicated cells were seeded at 3 × 105 cells per 60 mm dish. On day 1, the cells were treated with the indicated concentration of doxorubicin. On day 2, 24 hr after the treatment, some of the cells were harvested for quantification of p21 mRNA through RT-QPCR (B). On day 4, 72 hr after the treatment, the rest of the cells were harvested for quantification of collagen 1α1 (COL1A1) mRNA through RT-QPCR (A). (A),(B) The value of each mRNA in cells that were not treated with the drug is set to 1. (C) Immunoblot analysis of CREB3L1 in indicated cells. https://doi.org/10.7554/eLife.00090.004

Inasmuch as CREB3L1 was required for doxorubicin to induce expression of p21, a well-characterized inhibitor of the cell cycle (Sherr and Roberts, 1999), we determined whether CREB3L1 was also required for doxorubicin to inhibit cell proliferation. For both untransfected Huh7 cells and those transfected with the control shRNA (Huh7-shControl), doxorubicin completely blocked their proliferation at a concentration between 50 and 150 nM (Figure 3A). This concentration of doxorubicin also resulted in maximal cleavage of CREB3L1 in Huh7 cells (Figure 3B). For Huh7-shCREB3L1 cells, doxorubicin at concentrations up to 500 nM failed to block their proliferation (Figure 3A). These concentrations of doxorubicin were not enough to trigger apoptosis of Huh7 cells, which became apparent only when the cells were treated with 5 µM of the compound (Figure 3C). To rule out the off-target effects of the shRNA, we also transfected Huh7 cells with two distinct siRNA targeting regions of CREB3L1 that is different from that targeted by the shRNA. Transfection with these siRNA knocked down CREB3L1 mRNA by more than 90% (Figure 3D), and the treatment also rendered Huh7 cells more resistant to doxorubicin (Figure 3E).

Figure 3 Download asset Open asset CREB3L1 is required for doxorubicin to suppress proliferation of Huh7 cells. (A) On day 0, indicated cells were seeded at 1.5 × 105 cells per 60 mm dish. On day 1, they were treated with the indicated concentrations of doxorubicin. On day 3, 48 hr after the treatment, the cells were quantified to determine cell proliferation. The number of cells just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100%, respectively. (B) Huh7 cells treated with the indicated concentrations of doxorubicin were analyzed as described in Figure 1B. (C) On day 0, Huh7 cells were seeded at 4 × 105 cells per 60 mm dish. On day 1, cells were treated with the indicated concentrations of doxorubicin. On day 3, 48 hr after the treatment, cells were harvested to determine the percentage of the cells that underwent apoptosis through TUNEL assay. (D),(E) On day 0, Huh7 cells were seeded at 1 × 105 cells per 60 mm dish. On day 1, the cells were transfected with indicted siRNAs. On day 2, the cells were treated with indicated concentrations of doxorubicin. On day 4, 48 hr after the treatment, some of the cells were harvested for quantification of CREB3L1 mRNA by RT-QPCR (D), while the others were used for determination of cell proliferation as described in Figure 3A (E). (A),(C),(D),(E) Results are reported as mean ± S.E.M. of three independent experiments. https://doi.org/10.7554/eLife.00090.005

If proteolytic activation of CREB3L1 is required for doxorubicin to inhibit cell proliferation, then the amount of CREB3L1 expressed in cancer cells may determine their sensitivity to doxorubicin. To test this hypothesis, we analyzed SV589 cells, an immortalized line of human fibroblasts (Yamamoto et al., 1984), and MCF-7 cells, a line of human breast cancer cells (Soule et al., 1973). Compared to Huh7 cells, expression of CREB3L1 was higher in SV589 cells and lower in MCF-7 cells (Figure 4A). The sensitivity of the cells to growth inhibition by doxorubicin followed the order of CREB3L1 expression (Figure 4B). Similar to Huh7 cells, knockdown of CREB3L1 by two duplexes of siRNA targeting different regions of CREB3L1 in SV589 cells (Figure 4C) made them more resistant to doxorubicin (Figure 4D). Since MCF-7 cells expressed very little CREB3L1, we used these cells to study the effect of CREB3L1 overexpression on sensitivity to doxorubicin. We stably transfected MCF-7 cells with a plasmid encoding CREB3L1 and selected one clone of the cells with relatively low expression (MCF7/pCREB3L1(L); eightfold above parental cells) and another clone with high expression of CREB3L1 (MCF7/pCREB3L1(H); 300-fold above parental cells) (Figure 4E). The eightfold overexpression of CREB3L1 in MCF7/pCREB3L1(L) cells lowered the IC 50 for doxorubicin from 500 nM to 10 nM, and the 300-fold overexpression of CREB3L1 in MCF7/pCREB3L1(H) cells further reduced the IC 50 to ∼1 nM (Figure 4F). In this experiment, cells were treated with doxorubicin for 2 days. To determine the effect of CREB3L1 expression on proliferation of the cells treated with doxorubicin for a longer period of time, we incubated MCF-7 and MCF7/pCREB3L1(H) cells with 15 nM doxorubicin for 6 days. This treatment did not affect proliferation of MCF-7 cells, but markedly blocked proliferation of MCF7/pCREB3L1(H) cells, as determined by direct cell counting (Figure 4G) and by measurement of cellular DNA content (Figure 4H). Thus, CREB3L1 expression level is a key determinant of cellular sensitivity to doxorubicin.

Figure 4 Download asset Open asset Sensitivity of cancer cells to doxorubicin is correlated to their expression of CREB3L1. (A),(E) RT-QPCR quantification of CREB3L1 mRNA in indicated cells with its value in Huh7 (A) or MCF-7 cells (E) set to 1. (B),(F) Effect of doxorubicin on proliferation of the indicated cells was determined as described in Figure 3A. (C),(D) SV-589 cells were treated and analyzed as described in Figure 3D,E. (G),(H) On day 0, indicated cells were seeded at 1.5 × 105 cells per 60 mm dish. On day 1, the cells were treated with or without 15 nM doxorubicin. After incubation for the indicated period of time, cell proliferation was determined by direct counting of the cells (G) or by measurement of the amount of cellular DNA (H). (G),(H) The number of cells just before doxorubicin treatment at time 0 is set to one. (A–H) Results are reported as mean ± S.E.M. of three independent experiments. https://doi.org/10.7554/eLife.00090.006

We then determined the relationship between doxorubicin-induced cleavage of CREB3L1 and DNA breaks caused by inhibition of topoisomerase. Doxorubicin induced appearance of histone γH2AX, a marker for DNA breaks (Figure 5A, lane 2). However, this effect was unaffected by knockdown of CREB3L1 expression (Figure 5A, lane 5). This result suggests that cleavage of CREB3L1 does not lead to doxorubicin-induced DNA breaks. To investigate whether DNA breaks may lead to cleavage of CREB3L1, we examined etoposide, another chemotherapeutic drug that inhibits topoisomerase (Stähelin and von Wartburg, 1991). Unlike doxorubicin, etoposide failed to induce cleavage of CREB3L1 (Figure 5B, lane 3), even though etoposide was as effective as doxorubicin in causing DNA breaks (Figure 5A, lanes 2 and 3). Accordingly, knockdown of CREB3L1 in Huh7 cells did not increase their resistance to etoposide (Figure 5C), and overexpression of CREB3L1 in MCF7 cells also did not increase their sensitivity to the compound (Figure 5D). These results suggest that induction of CREB3L1 cleavage by doxorubicin is not related to its inhibitory activity towards topoisomerase. Besides etoposide, CREB3L1 was also not required for bleomycin or paclitaxel to inhibit cell growth, an observation suggesting that CREB3L1 may be specifically involved in doxorubicin-induced suppression of cell proliferation (Figure 5E–G).

Figure 5 Download asset Open asset CREB3L1 activation is independent from DNA breaks. (A) On day 0, indicated cells were seeded at 4 × 105 cells per 60 mm dish. On day 1, cells were treated with 500 nM doxorubicin or 500 nM etoposide. On day 2, 24 hr after the treatment, the cells were harvested for immunoblot analysis with antibodies reacting against γH2AX or actin. (B) Huh7 cells were seeded and treated as described in (A). On day 2, cells were separated into nuclear and membrane fractions and analyzed by immunoblot analysis as described in Figure 1B. (C) The effect of etoposide on proliferation of the indicated cells was determined as described in Figure 3A. (D)–(G) On day 0, indicated cells were seeded at 1.5 × 105 cells per 60 mm dish. On day 1, cells were treated with indicated concentrations of etoposide (D), doxorubicin (E), bleomycin (F), or paclitaxel (G). On day 3, 48 hr after the treatment, proliferation of the cells was determined by measurement of cellular DNA. The amount of DNA just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100%, respectively. (C)–(G) Results are reported as mean ± S.E.M. of three independent experiments. https://doi.org/10.7554/eLife.00090.007

RIP of membrane-bound transcription factors is known to be a signal transduction pathway that transfers signals from the ER to nucleus (Brown et al., 2000). Since ER is the site where most lipids are synthesized, we wondered whether doxorubicin may alter homeostasis of certain lipids that may result in cleavage of CREB3L1. Doxorubicin and daunorubicin, a chemotherapeutic drug derived from doxorubicin, were reported to induce de novo synthesis of ceramide (Bose et al., 1995; Liu et al., 2008), a class of lipid known to inhibit cell proliferation (Ogretmen and Hannun, 2004). De novo synthesis of ceramide is initiated with the condensation of palmitate and serine (Gault et al., 2010). This rate-limiting step in de novo synthesis of ceramide is catalyzed by serine palmitoyltransferase (SPT) (Linn et al., 2001). Ceramide synthesis also requires a reaction catalyzed by ceramide synthase (Mullen et al., 2012). We confirmed that doxorubicin stimulated ceramide synthesis by showing that treatment with the compound increased the amount of [14C]palmitate incorporated into ceramide in Huh7 cells (Figure 6A). Mass spectroscopy analysis revealed that doxorubicin primarily increased the amount of ceramide containing palmitate (16:0) as the amide-linked fatty acid (Figure 6B). In contrast to doxorubicin, etoposide failed to induce ceramide synthesis at a concentration at which cell proliferation was inhibited (Figure 6C).

Figure 6 Download asset Open asset Doxorubicin stimulates synthesis of ceramide. (A) On day 0, Huh7 cells were seeded at 2 × 105 per 60-mm dish. On day 1, the cells were treated with or without 500 nM doxorubicin. On day 2, 20 hr after the treatment, the cells were labeled with indicated concentrations of [14C]palmitate for additional 4 hr. Cell lipids were then extracted to determine the amount of [14C]palmitate incorporated into ceramide. *p=0.003; **p=0.02. (B) On day 0, Huh7 cells were seeded at 1.5 × 105 per 60-mm dish. On day 1, the cells were treated with or without 500 nM doxorubicin. On day 2, 24 hr after the treatment, the cells were harvested for ceramide analysis via LC-MS as described in ‘Materials and methods’. The amount of ceramide with indicated amide-linked fatty acids was presented. (C) Huh7 cells were treated with 500 nM doxorubicin or 1 µM etoposide, labeled with 3 µM [14C]palmitate, and analyzed as described in Figure 6A. (A)–(C) Results are reported as mean ± S.E.M. of triplicate incubations from a representative experiment. https://doi.org/10.7554/eLife.00090.008

To determine whether doxorubicin-induced ceramide synthesis is required to stimulate cleavage of CREB3L1, we treated Huh7 cells with myriocin, an inhibitor of SPT (Miyake et al., 1995). This treatment inhibited doxorubicin-induced cleavage of CREB3L1 (Figure 7A). Co-treatment with myriocin rendered the cells more resistant to doxorubicin (Figure 7B). Fumonisin B 1 , an inhibitor of ceramide synthase (Wang et al., 1991), also blocked doxorubicin-induced cleavage of CREB3L1 (Figure 7C). The observations that inhibition of two different enzymes involved in ceramide synthesis were both effective in blocking doxorubicin-induced cleavage of CREB3L1 strongly suggest that this cleavage is caused by increased synthesis of ceramide.

Figure 7 Download asset Open asset Doxorubicin-induced synthesis of ceramide stimulates cleavage of CREB3L1. (A),(C) On day 0, Huh7 cells were seeded at 4 × 105 per 60-mm dish. On day 1, the cells were treated with indicated concentrations of myriocin (A) or fumonisin B1 (C) for 2 hr, followed by co-incubation with 200 nM doxorubicin. On day 2, 24 hr after the doxorubicin treatment, cells were analyzed for cleavage of CREB3L1 by immunoblot analysis as described in Figure 1B. (B) Huh7 cells treated with or without 30 µM myriocin for 2 hr followed by co-treatment with doxorubicin were analyzed as described in Figure 3A. (D) Huh7 cells treated with 10 µM C 6 -ceramide for 3 hr were analyzed as described in Figure 6B. Results are reported as mean ± S.E.M. of triplicate incubations from a representative experiment. (E) Huh7 cells treated with indicated concentration of C 6 -ceramide for 24 hr were analyzed as described in Figure 1B. (F) Indicated cells treated with indicated concentration of C 6 -ceramide for 48 hr were analyzed as described in Figure 3A. (B),(F) Results are reported as mean ± S.E.M. of three independent experiments. https://doi.org/10.7554/eLife.00090.009