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Supplemental oxygen therapy (hyperoxia) in preterm babies with respiratory stress is associated with lung injury and the development of bronchopulmonary dysplasia. Endoplasmic reticulum (ER) homeostasis plays critical roles in maintaining cellular functions such as protein synthesis, folding, and secretion. Interruption of ER homeostasis causes ER stress and triggers the unfolded protein response, which can lead to apoptosis in persistently stressed cells. ERp57 is an ER protein and is associated with calreticulin and calnexin in protein glycosylation. In this study, we found hyperoxia downregulated ERp57 in neonatal rat lungs and cultured human endothelial cells. Transient transfection of ERp57 small interfering RNA significantly knocked down ERp57 expression and reduced hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was decreased from 26.8 to 9.9% in hyperoxia-exposed cells and from 37.8 to 5.0% in tunicamycin-treated cells. The activation of caspase-3 induced by hyperoxia or tunicamycin was diminished and immunoglobulin heavy chain-binding protein/ glucose-regulated protein 78-kDa (BiP/GRP78) induction was increased in ERp57 knockdown cells. Overexpression of ERp57 exacerbated hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was increased from 10.1 to 14.3% in hyperoxia-exposed cells and from 14.0 to 21.2% in tunicamycin-treated cells. Overexpression of ERp57 also augmented tunicamycin-induced caspase-3 activation and reduced BiP/GRP78 induction. Our results demonstrate that ERp57 can regulate apoptosis in human endothelial cells. It appears that knockdown of ERp57 confers cellular protection against hyperoxia- or tunicamycin-induced apoptosis by inhibition of caspase-3 activation and stimulation of BiP/GRP78 induction. endoplasmic reticulum stress; unfolded protein response; lung injury; bronchopulmonary dysplasia; immunoglobulin heavy chain-binding protein

Xu D, Perez RE, Rezaiekhaligh MH, Bourdi M, Truog WE. Knockdown of ERp57 increases BiP/GRP78 induction and protects against hyperoxia and tunicamycin-induced apoptosis. Am J Physiol Lung Cell Mol Physiol 297: L44-L5 1, 2009. First published May 1, 2009; doi: 10.1152/ajplung.90626.2008.-Supplemental oxygen therapy (hyperoxia) in preterm babies with respiratory stress is associated with lung injury and the development of bronchopulmonary dysplasia. Endoplasmic reticulum (ER) homeostasis plays critical roles in maintaining cellular functions such as protein synthesis, folding, and secretion. Interruption of ER homeostasis causes ER stress and triggers the unfolded protein response, which can lead to apoptosis in persistently stressed cells. ERp57 is an ER protein and is associated with caireticulin and calnexin in protein glycosylation. In this study, we found hyperoxia downregulated ERp57 in neonatal rat lungs and cultured human endothelial cells. Transient transfection of ERp57 small interfering RNA significantly knocked down ERp57 expression and reduced hyperoxiaor tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was decreased from 26.8 to 9.9% in hyperoxia-exposed cells and from 37.8 to 5.0% in tunicamycin-treated cells. The activation of caspase-3 induced by hyperoxia or tunicamycm was diminished and immunoglobulin heavy chain-binding proteinl glucose-regulated protein 78-kDa (BiP/GRP78) induction was increased in ERp57 knockdown cells. Overexpression of ERp57 exacerbated hyperoxiaor tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was increased from 10.1 to 14.3% in hyperoxia-exposed cells and from 14.0 to 2 1.2% in tunicamycintreated cells. Overexpression of ERp57 also augmented tunicamycininduced caspase-3 activation and reduced BiP/GRP78 induction. Our results demonstrate that ERp57 can regulate apoptosis in human endothelial cells. It appears that knockdown of ERp57 confers cellular protection against hyperoxiaor tunicamycin-induced apoptosis by inhibition of caspase-3 activation and stimulation of BiP/GRP78 induction. [ABSTRACT FROM AUTHOR]

Despite clinical use of immunosuppressive agents, the immunopathogenesis of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remains unclear. Src homology 3-binding protein 2 (SH3BP2), a scaffold protein, forms an immune signaling complex (signalosome) with 17 other proteins, including phospholipase Cγ2 (PLCγ2) and Rho-guanine nucleotide exchange factor VAV2 (VAV2). Bioinformatic analysis of human glomerular transcriptome (Nephrotic Syndrome Study Network cohort) revealed upregulated SH3BP2 in MCD and FSGS. The SH3BP2 signalosome score and downstream MyD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS. Immune pathway activation scores for Toll-like receptors, cytokine-cytokine receptor, and NOD-like receptors were increased in FSGS. Lower SH3BP2 signalosome score was associated with MCD, higher estimated glomerular filtration rate, and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6-fold and ~25-fold increases in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron-dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes, underscoring the importance of SH3BP2 in immune activation. SH3BP2 and its binding partners may determine the immune activation pathways resulting in podocyte injury leading to loss of the glomerular filtration barrier.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-β receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-β type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-β1 action. TGF-β1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-β1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-β1 induction of EMT. Mechanistically, a pathway involving increased TGF-β type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of small GTPase activation. In analysis of scratch wound assays, we found that small interfering RNA (siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-GTP level in HMVEC-LLy, demonstrating cross talk between both GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to VEGF is mediated via a pathway of podoplanin regulation of small GTPase activities, in particular Cdc42.