Vincent Duronio

Regulation of Bad Phosphorylation and Association with Bcl-xL by the MAPK/Erk Kinase

Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increased on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation was shown to be absolutely required for dissociation of Bad from Bcl-xL. These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function.

The life of a cell: apoptosis regulation by the PI3K/PKB pathway

The activation of PI3K (phosphoinositide 3-kinase) family members is a universal event in response to virtually all cytokines, growth factors and hormones. As a result of formation of PtdIns with an added phosphate at the 3 position of the inositol ring, activation of the protein kinases PDK1 (phosphoinositide-dependent kinase 1) and PKB (protein kinase B)/Akt occurs. The PI3K/PKB pathway impinges upon a remarkable array of intracellular events that influence either directly or indirectly whether or not a cell will undergo apoptosis. In this review, the many ways in which PI3K/PKB can control these processes are summarized. Not all of the events described will necessarily play a role in any one cell type, but a subset of these events is probably essential for the survival of every cell.

Downstream Signalling Events Regulated by Phosphatidylinositol 3-Kinase Activity

The phosphatidylinositol (PI) 3-kinase family of enzymes is now known to be regulated by several different upstream pathways in response to virtually all growth factors and cytokines. In the past few years, the phosphoinositides phosphorylated at the 3-OH position of the inositol ring have been shown to be lipid second messengers that may directly or indirectly regulate the activity of several different serine/threonine kinases. Consistent with the many different cellular events in which PI 3-kinase plays an important role, a diverse group of serine/threonine kinases are regulated downstream of PI 3-kinases, including protein kinase C (PKC) isoforms, p70 S6 kinase, and PKB/Akt. This review summarises studies done primarily in the past few years that have begun to unravel these targets of PI 3-kinase activity.

Targeted disruption of SHIP leads to Steel factor-induced degranulation of mast cells

To investigate the role of the src homology 2 (SH2)‐containing inositol 5′ phosphatase (SHIP) in growth factor‐mediated signalling, we compared Steel factor (SF)‐induced events in bone marrow‐derived mast cells (BMMCs) from SHIP−/− and SHIP+/+ littermates. We found SF alone stimulated massive degranulation from SHIP−/− but none from SHIP+/+ BMMCs. This SF‐induced degranulation, which was not due to higher c‐kit levels in SHIP−/− cells, correlated with higher intracellular calcium than that in SHIP+/+ cells and was dependent on the influx of extracellular calcium. Both this influx and subsequent degranulation were completely inhibited by PI‐3‐kinase inhibitors, indicating that SF‐induced activation of PI‐3‐kinase was upstream of extracellular calcium entry. A comparison of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) levels following SF stimulation of SHIP+/+ and SHIP−/− BMMCs suggested that SHIP restricted this entry by hydrolyzing PIP3. Although PI‐3‐kinase inhibitors blocked the release of intracellular calcium, implicating PIP3, and PLCγ‐2 was slightly more tyrosine phosphorylated in SHIP−/− cells, the increase in inositol‐1,4,5‐trisphosphate (IP3) and intracellular calcium levels were identical in SHIP−/− and SHIP+/+ BMMCs. These results suggest that SHIP prevents SF from triggering degranulation of normal BMMCs, and does so by hydrolyzing PIP3, which in turn limits extracellular calcium entry at a step after the release of intracellular calcium.

Ceramide‐1‐phosphate promotes cell survival through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway

In this report, we show for the first time that ceramide‐1‐phosphate (C1P) stimulates the phosphatidylinositol 3‐kinase (PI3‐K)/protein kinase B (PKB) pathway, which is a major mechanism whereby growth factors promote cell survival. Also, C1P induced IκB phosphorylation, and enhanced the DNA binding activity of the transcription factor NF‐κB. Apoptotic macrophages showed a marked reduction of Bcl‐XL levels, and this was prevented by C1P. These findings suggest that C1P blocks apoptosis, at least in part, by stimulating the PI3‐K/PKB/ NF‐κB pathway and maintaining the production of antiapoptotic Bcl‐XL. Based on these and our previous observations, we propose a working model for C1P in which inhibition of acid sphingomyelinase and the subsequent decrease in ceramide levels would allow cell signaling through stimulation of the PI3‐K/PKB pathway to promote cell survival.

Proliferation of Pulmonary Interstitial Fibroblasts Is Mediated by Transforming Growth Factor-β1-induced Release of Extracellular Fibroblast Growth Factor-2 and Phosphorylation of p38 MAPK and JNK

Idiopathic pulmonary fibrosis (IPF; a progressive lung disease) is characterized by parenchymal remodeling with enlarged air spaces called honeycomb cysts and palisades of fibroblasts called fibroblast foci. In IPF, lung epithelial cells covering honeycomb cysts and fibroblast foci aberrantly express the active conformation of the potent fibrogenic cytokine transforming growth factor-β1 (TGF-β1). Using explanted rat lung slices, we transfected alveolar epithelial cells with the retrovirus pMX containing a site-directed mutation in which Cys223 and Cys225 were substituted with serines, resulting in release of biologically active TGF-β1 and fibroblast proliferation and remodeling that resembled IPF. Fibroblasts obtained from transfected explants and in culture for 6 weeks incorporated 6.59 ± 1.55-fold more [3H]thymidine compared with control fibroblasts without transfection or fibroblasts obtained from transfected explants cultured with antibody to fibroblast growth factor-2 (FGF-2). Primary lung fibroblasts obtained from normal rat lungs cultured with TGF-β1 expressed increased levels of phosphorylated p38 MAPK and JNK, but not ERK1/2. The presence of TGF-β1 caused an immediate release of extracellular FGF-2 from primary pulmonary fibroblasts; and in the presence of anti-FGF-2 antibody, phosphorylated p38 MAPK and JNK were abrogated. TGF-β inhibits cell proliferation by suppression of c-Myc and induction of p15INK46, p21CIP1, or p27KIP. Fibroblasts cultured with TGF-β1 showed no regulation of c-Myc or induction of p15INK46, p21CIP1,or p27KIP. These findings suggest that pulmonary fibroblasts may not respond to the anti-proliferative effects of TGF-β1, but proliferate in response to TGF-β1 indirectly by the release of FGF-2, which induces phosphorylation of p38 MAPK and JNK.

What do we mean by the term “inflammation”? A contemporary basic science update for sports medicine

Most practicing sports medicine clinicians refer to the concept of “inflammation” many times a day when diagnosing and treating acute and overuse injuries. What is meant by this term? Is it a “good” or a “bad” process? The major advances in the understanding of inflammation in recent years are summarised, and some clinical implications of the contemporary model of inflammation are highlighted.

Excessive Apoptosis in Patellar Tendinopathy in Athletes

Background The pathogenesis of tendon overuse injuries is poorly understood. The histopathology underlying tendinopathy at various anatomical locations is similar and may reflect a common pathologic process. Hypothesis Apoptosis contributes to the pathophysiology in patellar tendinopathy. Study Design Case control study; Level of evidence, 3. Methods We compared biopsy specimens from the patellar tendon in patients with patellar tendinopathy diagnosed clinically and with typical magnetic resonance image findings with biopsy specimens from a control group without any previous or current knee complaints to suggest patellar tendinopathy. The presence of apoptosis was examined with immunohistochemical methods using a polyclonal antibody recognizing active caspase-3, confirmed by labeling DNA strand breaks (F7-26 antibody) and nuclear morphology (fragmentation and condensation). Results The number of apoptotic cells per unit area (4.5 mm2) was 0.91 ± 0.81 (SD) in tendinopathic samples and 0.21 ± 0.21 in controls (P = .026). Although the tendinopathic samples displayed increased cellularity (average 162.5 nuclei/mm 2 vs 98.9 nuclei/mm2), the apoptotic index was higher (0.42% vs 0.17%, P = .014). Conclusion Increased apoptotic cell death is a feature of patellar tendinosis. The role of apoptosis within the broader framework and time course of tendon overuse injury remains to be established.

PI(3,4,5)P3 and PI(3,4)P2 levels correlate with PKB/akt phosphorylation at Thr308 and Ser473, respectively; PI(3,4)P2 levels determine PKB activity

The PI3K-PKB pathway is an important and widely studied pathway in cell signaling. The enzyme activity of PI3K produces D-3 phosphoinositides, including the lipid second messengers PI(3,4,5)P3 and PI(3,4)P2. PI(3,4,5)P3 has been deemed to be the most important second messenger for triggering PKB phosphorylation. PKB has two regulatory phosphorylation sites, Thr308 and Ser473, both of which contribute to its full activity. The direct relationship between PI3K lipid products and PKB phosphorylation is still not entirely clear. Our previous study showed that PI(3,4)P2 has a specific role in contributing to PKB phosphorylation on Ser473 sites in mast cells. In this study, we used two strategies to further elucidate this question in a well-established B cell system. First, by SHIP overexpression, we examined PKB activation under conditions where PI(3,4,5)P3 accumulation is largely suppressed. Second, we used dose response of different forms of B-cell receptor ligands to manipulate the relative levels of PI(3,4,5)P3 and PI(3,4)P2. Our results demonstrate a close relationship between PI(3,4,5)P3 levels and Thr308 phosphorylation levels, and PI(3,4)P2 levels and Ser473 phosphorylation levels, respectively. Furthermore, overall PKB activity, primarily consisting of cytosolic enzyme, was dependent upon levels of PI(3,4)P2, while only membrane-associated PKB activity was dependent upon PI(3,4,5)P3 levels. We conclude that PI(3,4,5)P3 and PI(3,4)P2 have distinct roles in determining PKB phosphorylation and activity. Thus, when investigating PI3K-PKB pathways, the importance of both lipids must be considered.

Phosphatidylinositol 3,4,5-trisphosphate regulates Ca2+ entry via Btk in platelets and megakaryocytes without increasing phospholipase C activity

The role of phosphatidylinositol 3,4,5‐trisphosphate (PI3,4,5P3) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P3 was increased in platelets from mice deficient in the SH2 domain‐containing inositol 5‐phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cγ2 (PLCγ2) were unaltered in SHIP−/− platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca2+, maximal expression of P‐selectin, and potentiation of Ca2+ and aminophospholipid exposure to CRP in SHIP−/− platelets in the presence of Ca2+ (1 mM). Microinjection of PI3,4,5P3 into megakaryocytes caused a 3‐fold increase in Ca2+ in response to CRP, which was absent in X‐linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca2+ in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca2+ entry that involves PI3,4,5P3 and Btk, and which is independent of increased PLC activity.