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Expression of the nerve growth factor NGF is increased in pulmonary hypertension (PH). We have here studied whether oxidative stress and inflammation, two pathological conditions associated with transforming growth factor-β1 (TGF-β1) in PH, may trigger NGF secretion by pulmonary arterial (PA) cells. Effects of hydrogen peroxide (H2O2) and interleukin-1β (IL-1β) were investigated ex vivo on rat pulmonary arteries, as well as in vitro on human PA smooth muscle (hPASMC) or endothelial cells (hPAEC). TβRI expression was assessed by Western blotting. NGF PA secretion was assessed by ELISA after TGF-β1 blockade (anti-TGF-β1 siRNA, TGF-β1 blocking antibodies, TβRI kinase, p38 or Smad3 inhibitors). TβRI PA expression was evidenced by Western blotting both ex vivo and in vitro. H2O2 or IL-1β significantly increased NGF secretion by hPASMC and hPAEC, and this effect was significantly reduced when blocking TGF-β1 expression, binding to TβRI, TβRI activity, or signaling pathways. In conclusion, oxidative stress and inflammation may trigger TGF-β1 secretion by hPASMC and hPAEC. TGF-β1 may then act as an autocrine factor on these cells, increasing NGF secretion via TβRI activation. Since NGF and TGF-β1 are relevant growth factors involved in PA remodeling, such mechanisms may therefore be relevant to PH pathophysiology.

In intrapulmonary arteries (IPAs), mechanical forces due to blood flow control vessel tone, and these forces change during pulmonary hypertension (PH). Piezo1, a stretch-activated calcium channel, is a sensor of mechanical stress present in both endothelial cells (ECs) and smooth muscle cells (SMCs). The present study investigated the role of Piezo1 on IPA in the chronic hypoxia model of PH. Rats were raised in chronically hypoxic conditions for 1 (1W-CH, early stage) or 3 weeks (3W-CH, late-stage) of PH or in normoxic conditions (Nx). Immunofluorescence labeling and patch-clamping revealed the presence of Piezo1 in both ECs and SMCs. The Piezo1 agonist, Yoda1, induced an IPA contraction in Nx and 3W-CH. Conversely, Yoda1 induced an endothelial nitric oxide (eNOS) dependent relaxation in 1W-CH. In ECs, the Yoda1-mediated intracellular calcium concentration ([Ca2+]i) increase was greater in 1W-CH as compared to Nx. Yoda1 induced an EC hyperpolarization in 1W-CH. The eNOS levels were increased in 1W-CH IPA compared to Nx or 3W-CH PH and Yoda1 activated phosphorylation of Akt (Ser473) and eNOS (Ser1177). Thus, we demonstrated that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca2+]i, endothelial-dependent hyperpolarization, and Akt-eNOS pathway activation in the early stage of PH.

Study of the initial steps of the CD95-mediated signaling pathways is a field of intense research and a long list of actors has been described in the literature. Nonetheless, the dynamism of protein-protein interactions (PPIs) occurring in the presence or absence of its natural ligand, CD95L, and the cellular distribution where these PPIs take place render it difficult to predict what will be the cellular outcome associated with the receptor engagement. Accordingly, CD95 stimulation can trigger apoptosis, necroptosis, pyroptosis, or pro-inflammatory signaling pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphatidylinositol-3-kinase (PI3K). Recent data suggest that CD95 can also activate pattern recognition receptors (PRRs) known to sense damage-associated molecular patterns (DAMPs) such as DNA debris and dead cells. This activation might contribute to the pro-inflammatory role of CD95 and favor cancer development or severity of chronic inflammatory and auto-immune disorders. Herein, we discuss some of the molecular links that might connect the CD95 signaling to DAMP sensors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively kills various cancer cell types, but also leads to the activation of signaling pathways that favor resistance to cell death. Here, we investigated the as yet unknown roles of calcium signaling and autophagy regulatory proteins during TRAIL-induced cell death in leukemia cells. Taking advantage of the Gene Expression Profiling Interactive Analysis (GEPIA) project, we first found that leukemia patients present a unique TRAIL receptor gene expression pattern that may reflect their resistance to TRAIL. The exposure of NB4 acute promyelocytic leukemia cells to TRAIL induces intracellular Ca2+ influx through a calcium release-activated channel (CRAC)-dependent mechanism, leading to an anti-apoptotic response. Mechanistically, we showed that upon TRAIL treatment, two autophagy proteins, ATG7 and p62/SQSTM1, are recruited to the death-inducing signaling complex (DISC) and are essential for TRAIL-induced Ca2+ influx and cell death. Importantly, the treatment of NB4 cells with all-trans retinoic acid (ATRA) led to the upregulation of p62/SQSTM1 and caspase-8 and, when added prior to TRAIL stimulation, significantly enhanced DISC formation and the apoptosis induced by TRAIL. In addition to uncovering new pleiotropic roles for autophagy proteins in controlling the calcium response and apoptosis triggered by TRAIL, our results point to novel therapeutic strategies for sensitizing leukemia cells to TRAIL.

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.