Loss of CpFTSY Reduces Photosynthetic Performance and Affects Insertion of PsaC of PSI in Diatoms.

The chloroplast signal recognition particle (CpSRP) receptor (CpFTSY) is a component of the CpSRP pathway that post-translationally targets light-harvesting complex proteins (LHCPs) to the thylakoid membranes in plants and green algae containing chloroplasts derived from primary endosymbiosis. In plants, CpFTSY also plays a major role in the co-translational incorporation of chloroplast-encoded subunits of photosynthetic complexes into the thylakoids. This role has not been demonstrated in green algae. So far, its function in organisms with chloroplasts derived from secondary endosymbiotic events has not been elucidated. Here, we report the generation and characterization of mutants lacking CpFTSY in the diatom Phaeodactylum tricornutum. We found that this protein is not involved in inserting LHCPs into thylakoid membranes, indicating that the post-translational part of the CpSRP pathway is not active in this group of microalgae. The lack of CpFTSY caused an increased level of photoprotection, low electron transport rates, inefficient repair of photosystem II (PSII), reduced growth, a strong decline in the PSI subunit PsaC and upregulation of proteins that might compensate for a non-functional co-translational CpSRP pathway during light stress conditions. The phenotype was highly similar to the one described for diatoms lacking another component of the co-translational CpSRP pathway, the CpSRP54 protein. However, in contrast to cpsrp54 mutants, only one thylakoid membrane protein, PetD of the Cytb6f complex, was downregulated in cpftsy. Our results point to a minor role for CpFTSY in the co-translational CpSRP pathway, suggesting that other mechanisms may partially compensate for the effect of a disrupted CpSRP pathway.

The Copenhagen Actinic Keratosis Study (COAKS). A decentralised clinical trial to evaluate tolerability, safety and efficacy of daily field-directed topical treatment with cytosolic phospholipase A2 inhibitor, AVX001, in participants with actinic keratosis: protocol for a randomised controlled phase I/IIa trial.

INTRODUCTION: Actinic keratosis (AK) is the most common precancerous skin condition caused by long-term UV exposure. Given the high recurrence rate of 15%-53%, identifying safe and effective treatment options is warranted. AVX001, a cytosolic phospholipase A2α (cPLA2α) enzyme inhibitor, is a novel anti-inflammatory drug for field-directed, self-administered, topical therapy of AK. METHODS AND ANALYSIS: This study is a single-centre, randomised, vehicle-controlled, double-blind, parallel-group hybrid clinical trial in adults with multiple AK lesions Olsen grade 1 or 2. The hybrid design combines decentralised participant tasks and assessments with conventional in-clinic visits. Recruitment using targeted advertising on social media and eligibility prescreening are conducted via the Studies&Me online recruitment platform. Participants (n=60) are randomly assigned to 1 of 3 treatment arms: AVX001 gel 1%, AVX001 gel 3% or vehicle gel. The trial consists of a 4-week treatment period with daily field-directed topical application of the gel and an 8-week follow-up period. Participants attend in-clinic visits at baseline, week 4 and week 12. The remote participant trial tasks include questionnaires and upload of smartphone-obtained photos of the treated skin area using a study-specific web-based app. Both remote and in-clinic assessments of safety and efficacy will be performed. The primary objective is to evaluate the local tolerability of daily application of AVX001 gel (1% or 3%) compared with vehicle gel. Secondary objectives include safety, efficacy, dose-response efficacy relationship, treatment satisfaction and cosmetic outcome. Exploratory objectives include evaluations of tolerability and efficacy assessed by dermatologists using smartphone photos uploaded by participants, comparisons of in-clinic and remote assessments and assessment of AK-related skin changes by non-invasive optical imaging. ETHICS AND DISSEMINATION: Approved by the Ethics Committee of the Capital Region of Denmark (H-21018064) and the Danish Medicines Agency (2021032485). Results will be submitted for publication in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBERS: 2021-000934-32; NCT05164393.

Adipogenic Activity of Chemicals Used in Plastic Consumer Products.

Bisphenols and phthalates, chemicals frequently used in plastic products, promote obesity in cell and animal models. However, these well-known metabolism-disrupting chemicals (MDCs) represent only a minute fraction of all compounds found in plastics. To gain a comprehensive understanding of plastics as a source of exposure to MDCs, we characterized the chemicals present in 34 everyday products using nontarget high-resolution mass spectrometry and analyzed their joint adipogenic activities by high-content imaging. We detected 55,300 chemical features and tentatively identified 629 unique compounds, including 11 known MDCs. Importantly, the chemicals extracted from one-third of the products caused murine 3T3-L1 preadipocytes to proliferate, and differentiate into adipocytes, which were larger and contained more triglycerides than those treated with the reference compound rosiglitazone. Because the majority of plastic extracts did not activate the peroxisome proliferator-activated receptor γ and the glucocorticoid receptor, the adipogenic effects are mediated via other mechanisms and, thus, likely to be caused by unknown MDCs. Our study demonstrates that daily-use plastics contain potent mixtures of MDCs and can, therefore, be a relevant yet underestimated environmental factor contributing to obesity.

Logical and experimental modeling of cytokine and eicosanoid signaling in psoriatic keratinocytes.

Psoriasis is a chronic skin disease, in which immune cells and keratinocytes keep each other in a state of inflammation. It is believed that phospholipase A2 (PLA2)-dependent eicosanoid release plays a key role in this. T-helper (Th) 1-derived cytokines are established activators of phospholipases in keratinocytes, whereas Th17-derived cytokines have largely unknown effects. Logical model simulations describing the function of cytokine and eicosanoid signaling networks combined with experimental data suggest that Th17 cytokines stimulate proinflammatory cytokine expression in psoriatic keratinocytes via activation of cPLA2α-Prostaglandin E2-EP4 signaling, which could be suppressed using the anti-psoriatic calcipotriol. cPLA2α inhibition and calcipotriol distinctly regulate expression of key psoriatic genes, possibly offering therapeutic advantage when applied together. Model simulations additionally suggest EP4 and protein kinase cAMP-activated catalytic subunit alpha as drug targets that may restore a normal phenotype. Our work illustrates how the study of complex diseases can benefit from an integrated systems approach.

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

cPLA2α Enzyme Inhibition Attenuates Inflammation and Keratinocyte Proliferation.

As a regulator of cellular inflammation and proliferation, cytosolic phospholipase A2 α (cPLA2α) is a promising therapeutic target for psoriasis; indeed, the cPLA2α inhibitor AVX001 has shown efficacy against plaque psoriasis in a phase I/IIa clinical trial. To improve our understanding of the anti-psoriatic properties of AVX001, we sought to determine how the compound modulates inflammation and keratinocyte hyperproliferation, key characteristics of the psoriatic epidermis. We measured eicosanoid release from human peripheral blood mononuclear cells (PBMC) and immortalized keratinocytes (HaCaT) and studied proliferation in HaCaT grown as monolayers and stratified cultures. We demonstrated that inhibition of cPLA2α using AVX001 produced a balanced reduction of prostaglandins and leukotrienes; significantly limited prostaglandin E2 (PGE2) release from both PBMC and HaCaT in response to pro-inflammatory stimuli; attenuated growth factor-induced arachidonic acid and PGE2 release from HaCaT; and inhibited keratinocyte proliferation in the absence and presence of exogenous growth factors, as well as in stratified cultures. These data suggest that the anti-psoriatic properties of AVX001 could result from a combination of anti-inflammatory and anti-proliferative effects, probably due to reduced local eicosanoid availability.

Defining estrogenic mechanisms of bisphenol A analogs through high throughput microscopy-based contextual assays.

Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERß loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERß and act as ERß antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.

Type I PIPK-alpha regulates directed cell migration by modulating Rac1 plasma membrane targeting and activation.

Phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) is a critical regulator of cell migration, but the roles of the type I phosphatidylinositol-4-phosphate 5-kinases (PIPKIs), which synthesize PI4,5P(2), have yet to be fully defined in this process. In this study, we report that one kinase, PIPKI-alpha, is a novel upstream regulator of Rac1 that links activated integrins to the regulation of cell migration. We show that PIPKI-alpha controls integrin-induced translocation of Rac1 to the plasma membrane and thereby regulates Rac1 activation. Strikingly, this function is not shared with other PIPKI isoforms, is independent of catalytic activity, and requires physical interaction of PIPKI-alpha with the Rac1 polybasic domain. Consistent with its role in Rac1 activation, depletion of PIPKI-alpha causes pronounced defects in membrane ruffling, actin organization, and focal adhesion formation, and ultimately affects the directional persistence of migration. Thus, our study defines the role of PIPKI-alpha in cell migration and describes a new mechanism for the spatial regulation of Rac1 activity that is critical for cell migration.

Type I phosphatidylinositol phosphate kinase beta regulates focal adhesion disassembly by promoting beta1 integrin endocytosis.

Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated beta1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIbeta), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)), as a key regulator of this process. We found that knockdown of PIPKIbeta by RNA interference blocks the internalization of active beta1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIbeta blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIbeta as a novel regulator of focal adhesion disassembly and suggest that PIPKIbeta spatially regulates integrin endocytosis at adhesion sites to control cell migration.

SRC-3Delta4 mediates the interaction of EGFR with FAK to promote cell migration.

EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here, we identify SRC-3Delta4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Delta4 that promote the localization of SRC-3Delta4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, overexpression of SRC-3Delta4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Delta4 as a missing adaptor between EGFR and its downstream signaling molecule FAK to coordinately regulate EGF-induced cell migration. Our study also reveals that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.

Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element.

The expression of members of the Reg family of secreted lectin-like proteins is increased in response to stress, inflammation and damage in many tissues. In the stomach, Reg is located in enterochromaffin-like cells, where its expression is stimulated by the gastric hormone gastrin. We have examined the mechanisms by which gastrin stimulates expression of Reg-1. Deletional mutations of 2.1 to 0.1 kb of the rat Reg-1 promoter in a luciferase reporter vector were transiently transfected into gastric cancer AGS-G(R) cells. All promoter fragments tested showed similar relative increases in luciferase expression in response to gastrin (1 nM). The response to gastrin of the smallest (104 bp) construct was 4.2+/-0.4-fold over basal. These responses were reduced by Ro-32-0432, a protein kinase C inhibitor, by C3-transferase, a Clostridium botulinum toxin and a selective inhibitor of the Rho family GTPase RhoA, and by co-transfection with a dominant negative form of RhoA. Co-transfection with a constitutively active form of RhoA stimulated expression 11.6+/-1.7-fold over basal. Mutations through the 104 bp construct identified a C-rich element (C-79CCCTCCC-72) required for responses to gastrin, PKC (protein kinase C) and L63RhoA (the constitutively active form of human RhoA protein containing a glutamine-to-leucine substitution at position 63). EMSAs (electrophoretic-mobility-shift assays) using nuclear extracts of control and gastrin-stimulated AGS-G(R) cells and a probe spanning -86 to -64 bp revealed multiple binding proteins. There was no effect of gastrin on the pattern of binding. Supershift assays indicated that transcription factors Sp1 and Sp3 bound the C-rich sequence. We conclude that gastrin stimulates Reg expression via activation of PKC and RhoA, that a C-rich region (-79 to -72) is critical for the response and that Sp-family transcription factors bind to this region of the promoter.