Receptors required for CCN1/TNFα-induced apoptosis. Primary HSFs were subjected to various treatments as described below, and then treated with soluble CCN1, TNFα, or both for 4.5 h before scoring for apoptosis. (A) HSFs were pre-incubated with 100 μg/ml mAbs that neutralize TNFRI, TNFRII, or with anti-syndecan-4 polyclonal antibodies (100 μg/ml), or normal rabbit IgG for 30 min before treatment with CCN1 and TNFα. (B) Cells were incubated with various inhibitory mAbs (50 μg/ml) before treatment with CCN1 and/or TNFα. Antibodies used included GoH3 (anti-integrin α₆), P5D2 (anti-β₁), LM609 (anti-α_vβ₃), P1F6 (anti-α_vβ₅), and normal mouse IgG. (C) HSFs were treated with CCN1 or the mutants DM, TM, or D125A (4 μg/ml each), or a combination of DM and D125A, either with or without TNFα.

Induction of ROS accumulation by CCN1 and TNFα through distinct cellular sources and the requirement of ROS for apoptosis. HSFs were transfected with control siRNA (mixture of four irrelevant sequences), Rac1 siRNA, or Nox1 siRNA, or pretreated with the 5-lipoxygenase inhibitor MK886 (10 μM) or the mitochondrial complex I inhibitor rotenone (10 μM). Cells were then incubated with CCN1, CHX, and/or TNFα for 1 h before ROS detection or 4.5 h before apoptosis assays. The effects of siRNAs and inhibitors on ROS accumulation (A, C), and apoptosis (B, D) are shown. The efficacies of Rac1 and Nox1 siRNAs in transfected cells are shown by immunoblotting total cell lysates with anti-Rac1 and β-actin antibodies (E) or by RNA blotting probed with ³²P-labeled human Nox1 (revealing the 2.0 kb Nox1 mRNA) and GAPDH cDNA (F).

CCN1 induces ROS through its apoptotic receptors. HSFs were cultured on glass cover slips and incubated with CCN1 and/or TNFα before ROS detection by fluorescent microscopy after loading with H₂DCF-DA (5 μM in PBS). Ten randomly selected high-power fields were photographed for each sample, and the average fluorescence intensity per cell is presented in arbitrary units. (A) Cells were treated with CCN1 and/or TNFα for various times indicated. (B) Cells were pre-incubated (30 min) with 50 μg/ml each of the following antibodies before being treated with CCN1 for 2 h and assayed for ROS accumulation as above: P1F6 (anti-α_vβ₅), LM609 (anti-α_vβ₃), GoH3 (anti-α₆), rabbit polyclonal anti-syndecan-4 antibodies, and normal IgG as control. (C) Cells were treated with wild-type CCN1, DM, TM, D125A, or a combination of DM and D125A for 2 h.

Model for CCN1/TNFα synergism. Matrix signaling through CCN1 can override the antiapoptotic effects of NFκB, allowing TNFα to induce apoptosis (Figures 1, 2 and 3). Data presented in this paper support a model in which CCN1 binding to its receptors (integrins α_vβ₅, α₆β₁, and syndecan-4) results in elevated and prolonged ROS accumulation that is dependent on Rac1, 5-lipoxygenase, and mitochondria, leading to the biphasic activation of JNK in the presence of TNFα (Figures 3, 4 and 5). Others have shown that ROS inactivates phosphatases (Kamata et al, 2005), leading to sustained activation of JNK, which in turn triggers the degradation of c-FLIP (Chang et al, 2006) to allow the activation of caspases-8 by the TNFα-dependent cytoplasmic complex II (Micheau and Tschopp, 2003). Signals of caspases-8/10 are amplified through the mitochondria via Bax-mediated cytochrome c release and activation of caspase-9 and -3, leading to apoptosis.

Fibroblast adhesion to CCN proteins enables TNFα to induce apoptosis. Primary HSFs were treated as described and exposed to CCN1 and/or TNFα for 4.5 h where indicated. Apoptotic cells were scored as condensed cell nuclei. (A) HSFs were adhered on glass cover slips coated with purified CCN1, CCN2, CCN3, or FN in serum-free medium, and treated with TNFα or vehicle (control). Cells were subjected to TUNEL assay and counterstained with DAPI to assess apoptosis. (B) Percents of apoptotic nuclei in cells treated as in (A) were quantified, including those adhered on FN, LN, VN, Col. I, and FBN. (C) Cells adhered to matrix substrates as indicated were treated with soluble CCN1 and/or TNFα and scored for apoptosis. (D) HSFs were cultured for 2 days in medium with 10% FBS to allow deposition of endogenous ECM. After serum starvation, cells were treated with various concentrations of CCN1 as indicated with or without TNFα. In this entire study, all bar graphs show standard deviation of the mean of triplicate determinations. Each experiment in this study was repeated at least three times with similar results; one representative experiment is shown.

CCN1/TNFα-induced apoptosis is independent of NFκB signaling but completely dependent on ROS accumulation and the consequent biphasic JNK1/2 activation. HSFs were subjected to various treatments as described, and incubated with CCN1, TNFα, CHX, or a combination as indicated. (A) Fibroblasts were incubated in serum-free medium containing 1 μCi/ml of ³⁵S-methionine together with CCN1 and/or TNFα for 4 h, and incorporated radioactivity was measured as acid precipitable counts. CHX was included as control. (B) HSFs were pre-incubated with CHX for 30 min, and then treated for 4 h with CCN1, with or without TNFα. Apoptotic cells were counted after DAPI staining. (C) HSFs were incubated with CCN1 and/or TNFα for various times as indicated, and cell lysates were resolved on SDS–PAGE followed by immunoblotting with phospho-specific antibodies against S536 of NFκB-p65. Blots were stripped and re-probed with antibodies against total NFκB-p65. (D) NFκB-dependent transcription was assessed by transient transfection with a luciferase reporter construct driven by an NFκB-responsive sequence (Takada et al, 2004). Transfected cells were treated with CCN1 and/or TNFα for the indicated times, and luciferase activity in the cell lysates determined. The activities were normalized against transfection efficiency controls. (E) HSFs were pre-incubated for 30 min with either a cell-permeable JNK-inhibitory peptide (50 μM) or SP600125 (25 μM), and then treated with CCN1 and/or TNFα for 4 h before scoring for apoptosis. Where indicated, JNK inhibitors were added 3 h after CCN1/TNFα treatment. (F) HSFs were treated with CCN1 and/or TNFα for various times (10 min to 8 h) as indicated, and cell lysates were electrophoresed and immunoblotted with antibodies against dually phosphorylated JNK1/2 (T183/Y185). Blots were stripped and re-probed with antibodies recognizing total JNK1/2. (G) Cells were pre-incubated for 30 min with 10 mM NAC or 0.4 mM BHA and then treated with CCN1 and/or TNFα for 4 h before scoring for apoptosis. (H) Cells were either transfected with Nox1 siRNA for 48 h to downregulate Nox1, or pre-incubated for 30 min with Rac1 inhibitor NSC23766 (0.4 mM), MK886 (10 μM), or rotenone (10 μM), before treatment with CCN1/TNFα for 15 min or 5 h (see Figure 5 for further details). Cell lysates were resolved on SDS–PAGE and immunoblotted with antibodies against dually phosphorylated and total JNK1/2.

Generation of Ccn1^dm/dm mice and blunted TNFα-mediated apoptosis in vivo. (A) A gene targeting construct replaced the EcoRI–XmaI fragment of Ccn1 with a cDNA encoding Ccn1^dm. The recombined allele maintained the Ccn1 promoter and transcription start site and expresses the Ccn1^dm cDNA that preserved the 5′ and 3′ untranslated sequences. Thymidine kinase (tk) and PGK-neo were used as selectable markers in homologous recombination in ES cells. B, BamHI; X, XmaI; R, EcoRI; S, SphI. (B) DNA samples isolated from wild-type (WT) or Ccn1^dm/Ccn1 mice (+/−) were digested with SphI and probed with a BamHI/EcoRI fragment, yielding 6.3 (wild-type) and 5.7 (targeted) kb bands illustrated in (A). (C) Total RNA was isolated from MEFs, reverse-transcribed and the Ccn1 sequence amplified by PCR. The Ccn1^dm cDNA contains an engineered diagnostic Sph1 site, which is absent in the wild-type sequence. (D) MEFs from Ccn1^dm/dm mice and their wild-type littermates were serum-stimulated to induce synthesis of CCN1 while being labeled with ³⁵S-cysteine. Total cell lysates were passed through a heparin-sepharose column, and eluted with buffer containing varying concentrations of NaCl as indicated. Eluted proteins were immunoprecipitated with anti-CCN1 antibodies, resolved on SDS–PAGE and exposed to X-ray film. (E) ConA (20 mg/kg body weight) was delivered by tail-vein injection, and liver apoptosis analyzed 8 h thereafter by TUNEL assay (labeled with rhodamine). Numbers of apoptotic cells were counted in three randomly chosen fields and expressed as cells/1 mm² of tissue. (WT, n=5; Ccn1^dm/dm, n=8; ^*P<0.001). Histological sections are shown in (F). (G) Apoptosis was induced by subcutaneous injection of TNFα (400 ng in 50 μl). After 8 h, skin tissue from injection sites was collected, processed, and subjected to TUNEL assay. Numbers of apoptotic cells were counted as above. (WT, n=4; Ccn1^dm/dm, n=7; ^*P<0.001). Histological sections are shown in (H), with a higher magnification view shown in (J). (I) Liver tissue of ConA or PBS-treated WT or Ccn1^dm/dm mice were stained with anti-8-OHdG antibodies.