Neutral sphingomyelinase 2 activity and protein stability are modulated by phosphorylation of five conserved serines.

We previously presented that the neutral sphingomyelinase 2 (nSMase2) is the only SMase activated in human airway epithelial (HAE) cells following exposure to oxidative stress (ox-stress), yielding ceramide accumulation and thereby inducing apoptosis. Furthermore, we reported that nSMase2 is a phospho-protein in which the level of phosphorylation controls nSMase2 activation induced by ox-stress. Here we identify five specific serines that are phosphorylated in nSMase2 and demonstrate that their phosphorylation controls the nSMase2 activity upon ox-stress exposure in an interdependent manner. Furthermore, we show that the nSMase2 protein stability and thus its level of expression is also post-translationally regulated by these five serine phosphorylation sites. This study provides initial structure/function insights regarding nSMase2 phosphorylation sites and offers some new links for future studies aiming to fully elucidate nSMase2 regulatory machinery.

Systematic Review and Meta-Analysis of Nonoperative Platelet-Rich Plasma Shoulder Injections for Rotator Cuff Pathology.

BACKGROUND: Platelet-rich plasma (PRP) injections have been introduced to augment the recovery of patients with shoulder pathology. Although multiple studies have been published, no large-scale trials or meta-analyses have assessed the efficacy of nonoperative shoulder PRP injection. OBJECTIVE: To assess the efficacy of nonoperative PRP shoulder injection in rotator cuff pathology for pain as measured by the visual analog scale (VAS) and range of motion (ROM). DESIGN: Two authors independently screened the Medline and Cochrane databases to include prospective studies that reported VAS and ROM outcomes for nonoperative shoulder PRP injections for rotator cuff pathology. Study quality was assessed using the revised Cochrane Collaboration risk-of-bias tool and modified Downs and Black checklist. Subsequent meta-analysis was performed to determine the effect of nonoperative PRP injections on pain and ROM 3 to 12 months after intervention. RESULTS: Six studies met systematic review criteria. The included studies used different PRP formulations (concentration, leukocyte count), injection protocols (approach, injection number), and varied study designs. Three studies concluded that PRP provided no significant benefit for pain and ROM when compared to physical therapy. Within-group meta-analysis of six fairly heterogeneous studies (I2 77.8%) demonstrated a statistically significant (P < .001) improvement in pain 3 to 12 months after PRP injection. Within-group meta-analysis for four studies for shoulder flexion and abduction was found to be too heterogeneous to derive meaningful results. CONCLUSION: There is a limited quantity of high-quality studies that assess the efficacy of nonoperative PRP shoulder injection for pain and ROM. Systematic review of PRP injections did not demonstrate an improvement in pain or ROM compared to physical therapy. Although within-group meta-analysis of nonoperative PRP statistically showed that nonoperative PRP improved pain, the lack of adequate negative controls precludes the ability to conclude whether improvements were due to natural recovery or nonoperative PRP.

EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization.

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.