C/EBPβ LIP augments cell death by inducing osteoglycin

Many types of tumor cell are devoid of the extracellular matrix proteoglycan osteoglycin (Ogn), but its role in tumor biology is poorly studied. Here we show that RNAi of Ogn attenuates stress-triggered cell death, whereas its overexpression increases cell death. We found that the transcription factor C/EBPβ regulates the expression of Ogn. C/EBPβ is expressed as a full-length, active form (LAP) and as a truncated, dominant-negative form (LIP), and the LIP/LAP ratio is positively correlated with the extent of cell death under stress. For example, we reported that drug-resistant tumor cells lack LIP altogether, and its supplementation abolished their resistance to chemotherapy and to endoplasmic reticulum (ER) stress. Here we further show that elevated LIP/LAP ratio robustly increased Ogn expression and cell death under stress by modulating the mitogen-activated protein kinase/activator protein 1 pathway (MAPK/AP-1). Our findings suggest that LIP deficiency renders tumor cell resistant to ER stress by preventing the induction of Ogn.

Supplementary Figure 3 Inhibition of MAPK activity with specific inhibitors. Immunoblot of the indicated proteins in total cell extract of F10.9-4 cells pretreated with vehicle or doxycycline (Doxy), followed by vehicle or tunicamycin (Tm) in the presence or absence of the ERK1/2 inhibitor AZD6244, the JNK inhibitor JNK-IN-8 or the p38 inhibitor SCIO469.
Supplementary Figure 4 Effect of the MAPK inhibitors on activation of the MAPK/AP-1 axis. Immunoblot of the indicated proteins in total cell extract of F10.9-4 cells pretreated with vehicle or doxycycline (Doxy), followed by vehicle or tunicamycin (Tm) in the presence or absence of the ERK1/2 inhibitor U0126 a, the JNK inhibitor SP600125 b, or the p38 inhibitor SB203580 c.      Supplementary Figure 9 The pro-death effect of JNK is not mediated by c-Jun/ATF2 only. Crystal violet staining of F10.9-4 cells transfected with the indicated siRNA at time=0, treated with vehicle or doxycycline (Doxy) at time=24 h, followed by vehicle or tunicamycin (Tm) at time=48 h, in the presence or absence of the JNK inhibitor SP600125. **P<0.01, ***P<0.001.

Supplementary Figure 10
The UPR is not involved in LIP augmentation cell death. (a) Immunoblot of the indicated proteins in total cell extract of F10.9-4 cells pretreated with vehicle or doxycycline (Doxy), followed by vehicle or tunicamycin (Tm) for 24 h. (b) Immunoblot of the indicated proteins in total cell extract of F10.9-4 cells pretreated with vehicle or Doxy, followed by vehicle or Tm for the indicated times.

Supplementary Figure 14
Ogn expression is regulated by the MAPK/AP-1 axis. (a, c, e) qRT-PCR of Ogn mRNA isolated from F10.9-4 cells pretreated with vehicle or doxycycline (Doxy), followed by vehicle or tunicamycin (Tm) for the indicated times, in the presence or absence of the ERK1/2 inhibitor U0126 a, the JNK inhibitor SP600125 c or the p38 inhibitor SB203580 e. (b, d, f) Immunoblot of Ogn in total cell extracts of F10.9-4 cells treated as described in a, c, e. N.S., non-specific band.

a" b"
Supplementary Figure 15 Ogn is mainly regulated through the JNK/c-Jun axis. (a) Crystal violet staining of F10.9-4 cells transfected with the indicated siRNA at time=0, treated with vehicle or doxycycline (Doxy) at time=24 h, followed by vehicle or tunicamycin (Tm) at time=48 h, in the presence or absence of the JNK inhibitor SP600125. N=2, ns, not significant. (b and c) Crystal violet staining of F10.9-4 cells transfected with the indicated siRNA at time=0, treated with vehicle or Doxy at time=24 h, followed by vehicle or Tm at time=48 h. N=2, ns, not significant.