Abstract
Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2alpha). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2alpha phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRDelta6. Myotubes expressing PKRDelta6 showed no increase in eIF2alpha phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Inhibition of PKR prevented nuclear migration of NF-kappaB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-kappaB. Phosphorylation of PKR and eIF2alpha was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation.
Highlights
Changes in muscle protein synthesis and degradation rate in cancer cachexia probably arise from the presence of tumorderived factors, such as proteolysis-inducing factor (PIF),2 or increased production of endogenous factors, such as cytokines or Ang II
There was no effect of PIF on the uptake of labeled phenylalanine into myotubes, confirming that the effect was on protein synthesis, rather than
This study investigates the mechanism by which two factors PIF and Ang II, which are linked to cancer cachexia [6, 7], inhibit protein synthesis in murine myotubes and how in turn this may be related to an increased protein degradation through the ubiquitin-proteasome proteolytic pathway
Summary
Changes in muscle protein synthesis and degradation rate in cancer cachexia probably arise from the presence of tumorderived factors, such as proteolysis-inducing factor (PIF),2 or increased production of endogenous factors, such as cytokines or Ang II. Using an antibody that only detects PKR phosphorylated on Thr-446, Western blotting showed an increase in phosphorylation within 30 min of PIF can be considered as a stress factor in cancer cachexia, the treating myotubes with PIF (Fig. 4A) or within 2 h of treatment effect on eIF2␣ phosphorylation was determined.
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