A, DOX induces cardiomyocyte death. Adult mouse cardiomyocytes were treated with rhWISP1 at the indicated concentrations for 1 h prior to addition of DOX (1 μM for 24 h). Specificity of WISP1 activity was verified by incubating WISP1 with neutralizing antibodies or control IgG (10 μg/ml for 1 h) before addition. Mono and oligonucleosomal fragmented DNA in cytoplasmic extracts was quantified by an ELISA as described in ‘Materials and methods’. *P < 0.001 versus untreated, †P < 0.05, ††P < 0.001 versus DOX alone (n=12). B, DOX-mediated cell death by MTT cell viability assay. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1 h) prior to DOX addition (1 μM for 24 h). Cell viability was assessed by the MTT assay. *P < 0.001 versus untreated, †P < 0.01 versus DOX (n=12).

A, DOX promotes Bax translocation to mitochondria and stimulates cytochrome c release into cytoplasm. Cardiomyocytes were treated with 1 μM DOX for the indicated time periods. Mitochondrial and cytoplasmic extracts were analyzed for Bax and cytochrome c levels by immunoblotting. Purity of extracts was verified by GAPDH (cytoplasmic) and V-β (mitochondrial) expressions by immunoblotting (n=3). B, DOX-mediated cytochrome c release is inhibited by the pancaspase inhibitor. Cardiomyocytes were treated with Z-VED-fmk (50 μM in DMSO for 30 min) followed by DOX (1 μM) for 8 h. Z-FA-fmk served as a negative control. DMSO served as a solvent control. Cytochome c levels in cytoplasmic extracts were analyzed by a colorimetric assay. Purity of cytoplasmic extracts and equal loading were confirmed by immunoblotting for GAPDH (inset). *P < 0.001 versus untreated, † P < 0.01 versus DOX (n=6/group). C, DOX activates caspase-3. Cardiomyocytes were treated with either caspase-3 (Z-DEVD-fmk or pancaspase (Z-VED-fmk) inhibitor prior to DOX addition (1 μM for 8 h). Z-FA-fmk served as a negative control. DMSO served as a solvent control. Caspase-3 activity was determined using a fluorescent method. * P < 0.001 versus untreated, † P < 0.01 versus DOX (n=6/group). D, DOX activates PARP. Cardiomyocytes treated as in C were analyzed for cleaved PARP (89kDa) by immunoblotting using cleared whole cell homogenates. Un-cleaved PARP served as a loading control (n=3). Percent PARP cleavage is shown in the right hand panel. * P < 0.01 versus untreated, † P < at least 0.05 versus DOX+Z-FA-fmk. E, Caspase inhibition blunts DOX-mediated cardiomyocyte death. Cardiomyocytes treated as in C, but for 24 h, were analyzed for cell death by ELISA. * P < 0.001 versus untreated, † P < 0.001 versus DOX (n=12/group).

A, WISP1 induces PI3K activation. Cardiomyocytes were incubated with WISP1 (10 ng/ml for 1 h). p85α-associated PI3K activities were analyzed by ELISA as described in ‘Materials and methods’. The upper panel shows immunoblot analysis of the same samples with anti-p85 antibody. * P < 0.01 versus untreated (n=6). B, WISP1 activates Akt. Cardiomyocytes treated as in A were analyzed for Akt activation by immunoblotting using cleared cell lysates and pAkt (Thr308) or Akt antibodies. A representative of three independent experiments is shown. C, WISP1 activates Akt kinase activity. Cardiomyocytes treated as in A were analyzed for Akt kinase activity using immunecomplex kinase assays. GSK3 served as a substrate (n=3). D, E, F, Pharmacological inhibitors of Akt and PI3K, and forced expression of dnAkt or dnPI3Kp85 by adenoviral gene transfer block WISP1 induced Akt activation. Cardiomyocytes were transduced (MOI 100 for 24 h) with Ad.dnAkt (D), or dnPI3Kp85 (E), or pretreated with Akt1-X (2.5 μM in water for 1 h; D), Ly294002 (25 μM in DMSO for 1 h; F) or wortmannin (250 nM in DMSO for 1 h; F) prior to WISP1 addition (10 ng/ml for 1 h). Ad.GFP and DMSO served as respective controls. Expression of HA was analyzed by immunoblotting (D; αTubulin served as a loading control). Total and pAkt (Thr308) levels were analyzed by immunoblotting (n=3). G. Forced expression of dnAkt and dnPI3Kp85 inhibit the pro-survival effects of WISP1. Cardiomyocytes treated as in D and E were analyzed for cell death at 24 h by ELISA. *P < 0.001 v versus untreated, †P < 0.001 versus DOX, §P < 0.01 versus DOX+WISP1 (n=12/group). H, Forced expression of constitutively active Akt inhibits DOX-mediated cell death. Cardiomyocytes were transduced with myr.Akt or GFP (MOI 100 for 24 h) prior to DOX addition (1 μM for 24 h). Cell death was analyzed by ELISA. *P < 0.001 versus untreated, †P < 0.001 versus DOX+Ad.GFP (n=12/group). Quantitation and significance of the data in panels B, C, D, E and F are presented in Supplementary Fig. S5.

A, DOX induces p38 MAPK activation. Cardiomyocytes were treated with DOX (1 μM) for the indicated time periods. p38 MAPK activation was analyzed by immunoblotting using activation-specific antibodies (p-p38 MAPK Thr180/Tyr182) and cleared whole cell homogenates (n=3). B, DOX-mediated p38 MAPK activation is inhibited by SB 203580. Cardiomyocytes were treated with SB 203580 (SB; 1 μM in DMSO for 30 min) prior to DOX (1 μM for 30 min) addition, and then analyzed as in A (n=3). C, DOX induces JNK activation. Cardiomyocytes treated as in A were analyzed for JNK activation by immunoblotting using activation-specific antibodies and cleared whole cell homogenates (n=3). D, SP 600125 inhibits DOX-mediated JNK activation (pJNK Thr183/Tyr185). Cardiomyocytes were treated with SP 600125 (SP; 20 μM in DMSO for 30 min) prior to DOX (1 μM for 30 min) treatment, and then analyzed as in C (n=3). E, SB and SP attenuate DOX-induced Bax translocation to mitochondria. Cardiomyocytes were treated with SB and SP prior to DOX addition (1 μM for 8 h). Bax levels in mitochondria were analyzed by immunoblotting. V-β served as a loading and purity control. F, SB and SP attenuate DOX-induced cardiomyocyte death. Cardiomyocytes were treated with SB and SP prior to DOX addition (1 μM for 24 h). Cell death was analyzed as in 1A. *P < 0.001 versus untreated, †P < 0.001 versus DOX alone or DOX+DMSO (n=12). G, WISP1 inhibits DOX-mediated p38 MAPK activation. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1 h) prior to DOX (1 μM for 30 min) addition, and then analyzed as in A (n=3). H, WISP1 inhibits DOX-mediated JNK activation. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1 h) prior to DOX (1 μM for 30 min) addition, and then analyzed as in C (n=3). Quantitation and significance of the data in panels A, B, C, D, E, G and H are presented in Supplementary Fig. S7.

A, WISP1 induces Survivin expression. Cardiomyocytes were treated with WISP1 (10 ng/ml) for 3 h. Cleared whole cell homogenates were then analyzed for Survivin protein levels by immunoblotting. αTubulin served as a loading control. A representative of three independent experiments is shown. B, WISP1 induces Survivin expression via PI3K and Akt. Cardiomyocytes transduced with dnPI3Kp85 or dnAkt were treated with WISP1 (10 ng/ml for 3 h). Survivin expression was analyzed by immunoblotting (n=3). C, Forced expression of Survivin blunts DOX-induced cell death. Cardiomyocytes were transduced with wild-type (Ad.Survivin) or mutant (Ad.mSurvivin) Survivin (MOI 100 for 24 h; overexpression of wild-type and mutant Survivin is confirmed by immunoblotting, n=3) prior to DOX addition (1 μM for 24 h). Cell death was analyzed by ELISA. *P < 0.001 versus untreated, †P < 0.001 versus DOX, §P < 0.05 versus DOX (n=12/group).

While the cell death pathways regulated by DOX are highlighted in red, the pro-survival effects of WISP1 are shown in blue. Few of the many signal transduction pathways that still needs investigation are highlighted in purple. The results here demonstrate that the CCN family member WNT1-Inducible Signaling Pathway Protein-1 (WISP1) can significantly inhibit doxorubicin (DOX)-induced cardiomyocyte death via activation of diverse cell survival pathways. WISP1 inhibits DOX-mediated p53 activation, p38MAPK and JNK phosphorylation, mitochondrial translocation of Bax, release of cytochrome c, activation of caspase-3, and suppression of Bcl-2 and Bcl-xL expression in cardiomyocytes. Further, WISP1 induces PI3K-dependent Akt activation, PI3K-Akt-dependent Survivin expression, GSK3β phosphorylation, β-catenin nuclear translocation, and its own induction.

A, DOX induces p53 expression in a time-dependent manner. Cardiomyocytes were treated with DOX for the indicated periods, and p53 levels analyzed by immunoblotting of whole cell homogenates. αTubulin served as a loading control. Results from three independent experiments are summarized in the lower panel. *P < 0.001 versus control (C) at 3 h. B, DOX stimulates p53 DNA binding activity. Nuclear proteins extracted from cardiomyocytes treated as in A were analyzed for p53 DNA binding activity by EMSA using ³²P-labeled double stranded consensus p53 oligonucleotides (indicated by an arrow). Binding specificity of p53 DNA was shown by competition with 50-fold molar excess of unlabeled double-stranded mutant p53 oligonucleotide, Lane 1, and 50-fold molar excess of cold consensus p53 oligonucleotide, Lane 2. Lamin served as a loading control (n=3). C, DOX-induced p53 activation was quantified by ELISA. Nuclear extracts described in B (DOX for 3 h) were analyzed for p53 activation by ELISA, as described under ‘Materials and methods’. *P < 0.001 versus untreated (n=3/group). D, DOX stimulates p53-dependent reporter gene activity. Cardiomyocytes were transduced with Ad-p53-Luc (MOI 50), or Ad-MCS-Luc (MOI 50) as control; Ad.pRL-Luc (MOI 50) served as an internal control. After 24 h, the cells were treated with DOX (1 μM- for 7 h), and firefly and Renilla luciferase activities were determined. *P < 0.001 versus Untreated (n=6/group). E, Forced expression of mutant p53 inhibits DOX-mediated cardiomyocyte death. Cardiomyocytes transduced with adenoviral mutant p53 (MOI 100 for 24 h) were treated with DOX for 24 h. Ad.GFP served as a control. Cell death was analyzed by ELISA. *P < 0.001 versus untreated, †P < 0.01 versus DOX (n=12/group).

A, DOX inhibits Bcl-2. Cardiomyocytes were treated DOX (1 μM for 6 h). Bcl-2 expression was analyzed by immunoblotting using cleared whole cell homogenates. αTubulin served as a loading control. Results from three 3 independent experiments are summarized in the lower panel. *P < 0.01 versus untreated. B, DOX inhibits anti-apoptotic Bcl-xL, but induces pro-apoptotic Bcl-xS expression. Cardiomyocytes treated as in A were analyzed for Bcl-xL/xS expression by immunoblotting using cleared whole cell homogenates. αTubulin served as a loading control. Results from three 3 independent experiments are summarized in the lower panel. *P < at least 0.05 versus untreated. C, DOX inhibit Akt activation. Cardiomyocytes treated as in A, but for an hour, were analyzed for pAkt (Thr308) levels by immunoblotting using cleared whole cell homogenates and activation-specific antibodies. Both total Akt and αTubulin served as loading controls. Results from three 3 independent experiments are summarized in the lower panel. *P < 0.01 versus untreated. D, DOX inhibits pGSK3β levels. Cardiomyocytes treated as in A, but for two hours, were analyzed for total and pGSK3β (Ser9) levels by immunoblotting using cleared whole cell homogenates. Results from three 3 independent experiments are summarized in the lower panel. *P < 0.05 versus untreated. E, Proteasome inhibitor reverses DOX-induced Bcl-2 suppression. Cardiomyocytes were treated with the proteasomal inhibitor lactacystin (5 μM) for 30 min followed by the addition of DOX (1 μM for 6 h). Bcl-2 expression was then analyzed as in A. Results from three 3 independent experiments are summarized in the lower panel. *P < 0.001 versus untreated, †P < 0.01 versus DOX. F, Proteasome inhibitor fails to reverse DOX-induced Bcl-xL suppression. Cardiomyocytes treated as in E were analyzed for Bcl-xL expression by immunoblotting. Results from three 3 independent experiments are summarized in the lower panel *P < 0.05 versus untreated.

A, WISP1 attenuates DOX-mediated p53 activation. Cardiomyocytes were incubated with WISP1 (10 ng/ml for 1 h) prior to DOX addition (1 μM for 3 h). Nuclear proteins were extracted and analyzed for p53 activation by ELISA. *P < 0.001 versus untreated, †P < 0.01 versus DOX (n=3/group). B, WISP1 attenuates DOX stimulated p53-dependent reporter gene activity. Cardiomyocytes transduced with Ad-p53-Luc (MOI 50) were treated with WISP1 (10 ng/ml for 1h) followed by DOX (1 μM) for 7 h. Firefly and Renilla luciferase activities were then determined as described in ‘Materials and methods’. * P < 0.001 versus untreated, †P < 0.01 versus DOX (n=12/group). C, WISP1 inhibits DOX-induced Bax mitochondrial translocation. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1h) prior to DOX addition (1 μM for 8 h). Mitochondrial and cytoplasmic extracts were analyzed for Bax and cytochrome c levels by immunoblotting. Results from three independent experiments are summarized in the right hand panel. *P < 0.05 versus respective untreated, †P < 0.01 versus respective DOX treatment. D, WISP1 attenuates DOX stimulated cytochrome c release. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1 h) prior to DOX addition (1 μM for 8 h). Cytochome c levels in cytoplasmic extracts were analyzed by a colorimetric assay. Purity of cytoplasmic extracts and equal loading were confirmed by immunoblotting for GAPDH (inset). *P < 0.001 versus untreated, †P < 0.001 versus DOX (n=6/group). E, WISP1 inhibits DOX-induced caspase-3 activation. Cardiomyocytes treated as in D were analyzed for Caspase-3 activation using a fluorescent method. *P < 0.001 versus untreated, †P < 0.05 versus DOX (n=6/group). F, WISP1 inhibits DOX-mediated PARP activation. Cardiomyocytes treated as in D were analyzed for cleaved PARP (89kDa) by immunoblotting (n=3). Un-cleaved PARP served as a loading control. Percent PARP cleavage is shown in the right hand panel. *P < 0.001 versus untreated, †P < 0.001 versus WISP1.

A, WISP1 reverses DOX-induced suppression of Bcl-2. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1 h) prior to DOX addition (1 μM for 6 h). Bcl-2 expression was analyzed by immunoblotting. αTubulin served as a loading control. Results from three independent experiments are summarized in the lower panel. *P < 0.05 versus untreated, †P < 0.05 versus untreated, §P < 0.01 versus DOX. B, WISP1 reduces Bax (mito) to Bcl-2 (total) ratio. Results from panel A (Bcl-2) and 6C (mito, Bax, lane 1) were plotted as a ratio of mitochondrial Bax levels to total (whole cell) Bcl-2 levels following DOX and DOX+WISP1 treatment. *P < 0.001 versus untreated, †P < 0.01 versus DOX (n=3). C, WISP1 reverses DOX-induced suppression of Bcl-2 expression in mitochondrial fraction. Cardiomyocytes were treated as in A, but were analyzed for Bcl-2 levels immunoblotting using mitochondrial fraction. V-β served as a loading and purity control. Ratios of mitochondrial Bax to mitochondrial bcl-2 levels are shown in the lower panel. *P < 0.001 versus untreated, †P < 0.01 versus DOX (n=3). D, WISP1 reverses DOX-induced suppression of Bcl-xL. Cardiomyocytes were treated with WISP1 (10 ng/ml for 1 h) prior to DOX addition (1 μM for 6 h). Bcl-x expression was analyzed by immunoblotting. αTubulin served as a loading control. Results from three independent experiments are summarized in the lower panel. *P < 0.01 versus untreated, †P < at least 0.05 versus DOX, E, WISP1 reduces Bax (mito) to Bcl-xL (total) ratio. Results from panel D (Bcl-xL) and 6C (mito, Bax, lane 1) were plotted as a ratio of mitochondrial Bax levels to total (whole cell) Bcl-xL levels following DOX and DOX+WISP1 treatment. *P < 0.001 versus untreated, †P < 0.001 versus DOX (n=3). F, WISP1 reverses DOX-induced suppression of Bcl-xL expression in mitochondrial fraction. Cardiomyocytes were treated as in D, but were analyzed for Bcl-2 levels immunoblotting using mitochondrial fraction. V-β served as a loading and purity control. Ratios of mitochondrial Bax to mitochondrial Bcl-xL levels are shown in the lower panel. *P < 0.001 versus untreated, †P < 0.05 versus DOX (n=3). G, Forced expression of wild-type Bcl-2 or Bcl-xL inhibits DOX-induced cell death. Cardiomyocytes transfected with Ad.Bcl-2 or Ad.Bcl-xL (MOI 100 for 24 h; expression levels of Bcl-2 and Bcl-xL following transduction are shown in panel H, n=3) was treated with DOX (1 μM for 24 h). Cell death was analyzed by ELISA. *P < 0.001 versus untreated, †P < at least 0.05 versus DOX+Ad.GFP (n=12/group).

A, WISP1 induces GSK3β phosphorylation. Cardiomyocytes were treated with WISP1 (10 ng/ml), and at the indicated time periods, whole cell homogenates were prepared and analyzed for total and phospho-GSK3β (Ser9) by immunoblotting (n=3). B, WISP1-mediated GSK3β phosphorylation is inhibited by SB-216763 and LiCl. Cardiomyocytes were treated with a GSK3β-specific inhibitor SB-216763 (1 nM in DMSO for 15 min) or LiCl (30 mM for 15 min) prior to the addition of WISP1 (10 ng/ml for 1h). Cleared whole cell homogenates were analyzed for total and phospho-GSK3β (Ser9) as in A (n=3). C, D, WISP1 induces GSK3β phosphorylation via PI3K and Akt. Cardiomyocytes were either transduced (C) with adenoviral vectors for dnPI3Kp85 and dnAkt (100 MOI for 24 h), or treated (D) with the PI3K inhibitor Ly294002 (25 μM in DMSO for 1 h) or Akt inhibitor Akti-X (2.5 μM in water for 1 h) prior to WISP1 addition (10 ng/ml for 1h). Cleared whole cell homogenates were analyzed for total and phospho-GSK3β (Ser9) as in A (n=3). E, WISP1 induces β-catenin nuclear translocation. Cardiomyocytes treated as in A were analyzed for β-catenin by immunoblotting using nuclear protein extracts (n=3). Lamin served as loading control. F, WISP1 induces its own expression. Cardiomyocytes were treated with WISP1 for the indicated time periods. Specificity of WISP1 was verified by incubating WISP1 (10 ng/ml) with neutralizing antibodies or control IgG (10 μg/ml for 1 h) before addition. WISP1 mRNA expression was analyzed by Northern blotting (n=3). 28S rRNA served as a loading control. G, WISP1 induces WISP1 protein expression. Cardiomyocytes treated as in F with WISP1, but for up to 24 h, were analyzed for WISP1 protein expression by immunoblotting using cleared whole cell homogenates (n=3). αTubulin served as a loading control. Quantitation and significance of the data in panels A-G are presented in Supplementary Fig. S10.