Temporal analysis of PI-3kinase and MAPK signal transduction during skeletal muscle overload

Skeletal muscles can adapt to increased mechanical forces (or loading) by increasing the size and strength of the muscle. Knowledge of the molecular mechanisms by which muscle responds to increased loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. The objective of this research was to examine the temporal associations between the activation of specific signaling pathway intermediates and their potential upstream regulator(s) in response to increased muscle loading. Previous work has demonstrated that focal adhesion kinase (FAK) activity is increased in overloaded hypertrophying skeletal muscle. Thus FAK is a candidate for transducing the loading stimulus in skeletal muscle, potentially by activating phosphatidylinositol 3-kinase (PI3K) and members of the mitogen-activated protein kinase (MAPK) family. However, it was unknown if muscle overload would result in activation of PI3K or the MAPKs. Thus, this work seeks to characterized the temporal response of (1) MAPK phosphorylation (including Erk 2, p38 MAPK and JNK), (2) PI3K activity, and (3) FAK tyrosine phosphorylation in response to 24 hours of compensatory overload in the rat soleus and plantaris muscles. In both muscles, overload resulted in transient Increases in the phosphorylation state of Erk2 and JNK, which peaked within the first hour of overload and returned to baseline thereafter. In contrast, p38 MAPK phosphorylation remained elevated throughout the entire 24-hour overload period. Moreover, overload increased PI3K activity only, in the plantaris and only at 12 hours. Moreover, 24 hours of overload induced a significant increase in total protein content in the plantaris but not the soleus. Thus an increase in total muscle protein content within the 24-hour loading period was observed only in muscle exhibiting increased PI3K activity. Surprisingly, FAK tyrosine phosphorylation was not increased during the overload period in either muscle, indicating that PI3K activation and increased MAPK phosphorylation were independent of increased FAK tyrosine phosphorylation. In summary, increased PI3K activity and sustained elevation of p38 MAPK phosphorylation were associated with muscle overload, identifying these pathways as potential mediators of the early hypertrophic response to skeletal muscle overload. This suggests that stimuli or mechanisms that activate these pathways may reduce/minimize muscle wasting and frailty. ^

Relevant drug to drug interactions between itraconazole and non-nucleoside reverse transcriptase inhibitors in HIV-infected patients on maintenance therapy for disseminated histoplasmosis

Objective. Itraconazole is recommended life-long for preventing relapse of disseminated histoplasmosis in HIV-infected patients. I sought to determine if serum itraconazole levels are affected by the type of Highly Active Anti-Retroviral Therapy (NNRTI or PI) being taken concomitantly to treat HIV. ^ Design. Retrospective cohort. ^ Methods. De-identified data were used from an IRB-approved parent study which identified patients on HAART and maintenance itraconazole for confirmed disseminated histoplasmosis between January 2003 and December 2006. Available itraconazole blood levels were abstracted as well as medications taken by each patient at the time of the blood tests. Mean itraconazole levels were compared using the student's t-test. ^ Results. 11 patients met study criteria. Patient characteristics were: median age 36, 91% men, 18% white, 18% black, 55% Hispanic and 9% Asians, median CD4 cell count 120 cells/mm3. 14 blood levels were available for analysis—8 on PI, 4 on NNRTI and 2 on both. 8/8 itraconazole levels obtained while taking concomitant PI were therapeutic (>0.4 μg/mL) in contrast to 0/4 obtained while taking NNRTI. Two patients switched from NNRTI to PI and reached therapeutic levels. Mean levels on NNRTI (0.05 μg/mL, s.d. 0.0) and on PI (2.45 μg/mL, s.d. 0.21) for these two patients were compared via a paired t-test (t = 16.00, d.f. = 1, P = 0.04). Remaining patient levels were compared using an unpaired t-test. Mean itraconazole on concomitant PI (n = 6) was 1.37 μg/mL (s.d. 0.74), while the mean on concomitant NNRTI was 0.05 μg/mL (s.d. 0.0), t = 2.39, d.f. = 6, P = 0.05. ^ Conclusions. Co-administration of NNRTI and itraconazole results in significant decreases in itraconazole blood levels, likely by inducing the CYP3A4 enzyme system. Itraconazole drug levels should be monitored in patients on concomitant NNRTI. PI-based HAART may be preferred over NNRTI-based HAART when using itraconazole to treat HIV-infected patients with disseminated histoplasmosis. ^

NHERF1 recruits the tumor suppressors PTEN and PHLPP to synergistically inhibit the PI-3K pathway

Glioblastoma multiforme is the most common form of brain cancer that presents patients with a poor prognosis that has remained unchanged over the past few decades. The tumor suppressor phosphatase PTEN antagonizes one of the major oncogenic pathways involved in the progression of glioblastoma, and is frequently deleted in this cancer type. Contrary to our expectations, we found that most glioblastoma cells expressing endogenous PTEN also harbor basal PI-3K/AKT activation mainly attributable to impaired PTEN membrane localization. This alteration correlated with a shift of the adaptor protein NHERF1, which contributes to PTEN membrane recruitment in normal cells, from the membrane to the cytoplasm. In cells expressing membrane-localized NHERF1, only simultaneous PTEN and NHERF1 depletion achieved AKT activation, suggesting the involvement of additional PI-3K/AKT suppressor regulated by NHERF1. We identified these novel interactors of NHERF1 as the PHLPP1 and PHLPP2 phosphatases. ^ NHERF1 directly interacted and recruited both PHLPP proteins to the membrane and, through both NHERF1 PDZ domains, assembled ternary complexes consisting of PTEN-NHERF1-PHLPP. Only simultaneous depletion of PTEN and PHLPP1 significantly activated AKT and increased proliferation in cells with membrane-localized NHERF1. Analysis of glioblastoma human tumors revealed frequent loss of membrane-localized NHERF1. On the other hand, targeting of NHERF1 to the membrane achieved suppression of AKT and cell proliferation. Our findings reveal a novel mechanism for PI-3K/AKT regulation by the synergistic cooperation between two important tumor suppressors, PTEN and PHLPP, via the scaffold protein NHERF1. ^