Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

A new Caenorhabditis elegans model to study copper toxicity in Wilson disease.

Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a copper (Cu) transporting ATPase whose trafficking from the Golgi to endo-lysosomal compartments drives sequestration of excess Cu and its further excretion from hepatocytes into the bile. Loss of ATP7B function leads to toxic Cu overload in the liver and subsequently in the brain, causing fatal hepatic and neurological abnormalities. The limitations of existing WD therapies call for the development of new therapeutic approaches, which require an amenable animal model system for screening and validation of drugs and molecular targets. To achieve this objective, we generated a mutant Caenorhabditis elegans strain with a substitution of a conserved histidine (H828Q) in the ATP7B ortholog cua-1 corresponding to the most common ATP7B variant (H1069Q) that causes WD. cua-1 mutant animals exhibited very poor resistance to Cu compared to the wild-type strain. This manifested in a strong delay in larval development, a shorter lifespan, impaired motility, oxidative stress pathway activation, and mitochondrial damage. In addition, morphological analysis revealed several neuronal abnormalities in cua-1 mutant animals exposed to Cu. Further investigation suggested that mutant CUA-1 is retained and degraded in the endoplasmic reticulum, similarly to human ATP7B-H1069Q. As a consequence, the mutant protein does not allow animals to counteract Cu toxicity. Notably, pharmacological correctors of ATP7B-H1069Q reduced Cu toxicity in cua-1 mutants indicating that similar pathogenic molecular pathways might be activated by the H/Q substitution and, therefore, targeted for rescue of ATP7B/CUA-1 function. Taken together, our findings suggest that the newly generated cua-1 mutant strain represents an excellent model for Cu toxicity studies in WD.

Deletion of IFT20 exclusively in the RPE ablates primary cilia and leads to retinal degeneration.

Vision impairment places a serious burden on the aging society, affecting the lives of millions of people. Many retinal diseases are of genetic origin, of which over 50% are due to mutations in cilia-associated genes. Most research on retinal degeneration has focused on the ciliated photoreceptor cells of the retina. However, the contribution of primary cilia in other ocular cell types has largely been ignored. The retinal pigment epithelium (RPE) is a monolayer epithelium at the back of the eye intricately associated with photoreceptors and essential for visual function. It is already known that primary cilia in the RPE are critical for its development and maturation; however, it remains unclear whether this affects RPE function and retinal tissue homeostasis. We generated a conditional knockout mouse model, in which IFT20 is exclusively deleted in the RPE, ablating primary cilia. This leads to defective RPE function, followed by photoreceptor degeneration and, ultimately, vision impairment. Transcriptomic analysis offers insights into mechanisms underlying pathogenic changes, which include transcripts related to epithelial homeostasis, the visual cycle, and phagocytosis. Due to the loss of cilia exclusively in the RPE, this mouse model enables us to tease out the functional role of RPE cilia and their contribution to retinal degeneration, providing a powerful tool for basic and translational research in syndromic and non-syndromic retinal degeneration. Non-ciliary mechanisms of IFT20 in the RPE may also contribute to pathogenesis and cannot be excluded, especially considering the increasing evidence of non-ciliary functions of ciliary proteins.

The SGLT2 inhibitor dapagliflozin improves kidney function in glycogen storage disease XI.

Glycogen storage disease XI, also known as Fanconi-Bickel syndrome (FBS), is a rare autosomal recessive disorder caused by mutations in the SLC2A2 gene that encodes the glucose-facilitated transporter type 2 (GLUT2). Patients develop a life-threatening renal proximal tubule dysfunction for which no treatment is available apart from electrolyte replacement. To investigate the renal pathogenesis of FBS, SLC2A2 expression was ablated in mouse kidney and HK-2 proximal tubule cells. GLUT2Pax8Cre+ mice developed time-dependent glycogen accumulation in proximal tubule cells and recapitulated the renal Fanconi phenotype seen in patients. In vitro suppression of GLUT2 impaired lysosomal autophagy as shown by transcriptomic and biochemical analysis. However, this effect was reversed by exposure to a low glucose concentration, suggesting that GLUT2 facilitates the homeostasis of key cellular pathways in proximal tubule cells by preventing glucose toxicity. To investigate whether targeting proximal tubule glucose influx can limit glycogen accumulation and correct symptoms in vivo, we treated mice with the selective SGLT2 inhibitor dapagliflozin. Dapagliflozin reduced glycogen accumulation and improved metabolic acidosis and phosphaturia in the animals by normalizing the expression of Napi2a and NHE3 transporters. In addition, in a patient with FBS, dapagliflozin was safe, improved serum potassium and phosphate concentrations, and reduced glycogen content in urinary shed cells. Overall, this study provides proof of concept for dapagliflozin as a potentially suitable therapy for FBS.

TFEB and TFE3 control glucose homeostasis by regulating insulin gene expression.

To fulfill their function, pancreatic beta cells require precise nutrient-sensing mechanisms that control insulin production. Transcription factor EB (TFEB) and its homolog TFE3 have emerged as crucial regulators of the adaptive response of cell metabolism to environmental cues. Here, we show that TFEB and TFE3 regulate beta-cell function and insulin gene expression in response to variations in nutrient availability. We found that nutrient deprivation in beta cells promoted TFEB/TFE3 activation, which resulted in suppression of insulin gene expression. TFEB overexpression was sufficient to inhibit insulin transcription, whereas beta cells depleted of both TFEB and TFE3 failed to suppress insulin gene expression in response to amino acid deprivation. Interestingly, ChIP-seq analysis showed binding of TFEB to super-enhancer regions that regulate insulin transcription. Conditional, beta-cell-specific, Tfeb-overexpressing, and Tfeb/Tfe3 double-KO mice showed severe alteration of insulin transcription, secretion, and glucose tolerance, indicating that TFEB and TFE3 are important physiological mediators of pancreatic function. Our findings reveal a nutrient-controlled transcriptional mechanism that regulates insulin production, thus playing a key role in glucose homeostasis at both cellular and organismal levels.

TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.

Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

EGR1 drives cell proliferation by directly stimulating TFEB transcription in response to starvation.

The stress-responsive transcription factor EB (TFEB) is a master controller of lysosomal biogenesis and autophagy and plays a major role in several cancer-associated diseases. TFEB is regulated at the posttranslational level by the nutrient-sensitive kinase complex mTORC1. However, little is known about the regulation of TFEB transcription. Here, through integrative genomic approaches, we identify the immediate-early gene EGR1 as a positive transcriptional regulator of TFEB expression in human cells and demonstrate that, in the absence of EGR1, TFEB-mediated transcriptional response to starvation is impaired. Remarkably, both genetic and pharmacological inhibition of EGR1, using the MEK1/2 inhibitor Trametinib, significantly reduced the proliferation of 2D and 3D cultures of cells displaying constitutive activation of TFEB, including those from a patient with Birt-Hogg-Dubé (BHD) syndrome, a TFEB-driven inherited cancer condition. Overall, we uncover an additional layer of TFEB regulation consisting in modulating its transcription via EGR1 and propose that interfering with the EGR1-TFEB axis may represent a therapeutic strategy to counteract constitutive TFEB activation in cancer-associated conditions.

Analysis of inhibitors of the anoctamin-1 chloride channel (transmembrane member 16A, TMEM16A) reveals indirect mechanisms involving alterations in calcium signalling.

BACKGROUND AND PURPOSE: Pharmacological inhibitors of TMEM16A (ANO1), a Ca2+ -activated Cl- channel, are important tools of research and possible therapeutic agents acting on smooth muscle, airway epithelia and cancer cells. We tested a panel of TMEM16A inhibitors, including CaCCinh -A01, niclosamide, MONNA, Ani9 and niflumic acid, to evaluate their possible effect on intracellular Ca2+ . EXPERIMENTAL APPROACH: We recorded cytosolic Ca2+ increase elicited with UTP, ionomycin or IP3 uncaging. KEY RESULTS: Unexpectedly, we found that all compounds, except for Ani9, markedly decreased intracellular Ca2+ elevation induced by stimuli acting on intracellular Ca2+ stores. These effects were similarly observed in cells with and without TMEM16A expression. We investigated in more detail the mechanism of action of niclosamide and CaCCinh -A01. Acute addition of niclosamide directly increased intracellular Ca2+ , an activity consistent with inhibition of the SERCA pump. In contrast to niclosamide, CaCCinh -A01 did not elevate intracellular Ca2+ , thus implying a different mechanism of action, possibly a block of inositol triphosphate receptors. CONCLUSIONS AND IMPLICATIONS: Most TMEM16A inhibitors are endowed with indirect effects mediated by alteration of intracellular Ca2+ handling, which may in part preclude their use as TMEM16A research tools.

Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.

Airway surface hyperviscosity and defective mucociliary transport by IL-17/TNF-α are corrected by ß-adrenergic stimulus.

The fluid covering the surface of airway epithelia represents a first barrier against pathogens. The chemical and physical properties of the airway surface fluid are controlled by the activity of ion channels and transporters. In cystic fibrosis (CF), loss of CFTR chloride channel function causes airway surface dehydration, bacterial infection, and inflammation. We investigated the effects of IL-17A plus TNF-α, 2 cytokines with relevant roles in CF and other chronic lung diseases. Transcriptome analysis revealed a profound change with upregulation of several genes involved in ion transport, antibacterial defense, and neutrophil recruitment. At the functional level, bronchial epithelia treated in vitro with the cytokine combination showed upregulation of ENaC channel, ATP12A proton pump, ADRB2 ß-adrenergic receptor, and SLC26A4 anion exchanger. The overall result of IL-17A/TNF-α treatment was hyperviscosity of the airway surface, as demonstrated by fluorescence recovery after photobleaching (FRAP) experiments. Importantly, stimulation with a ß-adrenergic agonist switched airway surface to a low-viscosity state in non-CF but not in CF epithelia. Our study suggests that CF lung disease is sustained by a vicious cycle in which epithelia cannot exit from the hyperviscous state, thus perpetuating the proinflammatory airway surface condition.

The differential expression of PilY1 proteins by the HsfBA phosphorelay allows twitching motility in the absence of exopolysaccharides.

Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions.

ß-catenin perturbations control differentiation programs in mouse embryonic stem cells.

The Wnt/ß-catenin pathway is involved in development, cancer, and embryonic stem cell (ESC) maintenance; its dual role in stem cell self-renewal and differentiation is still controversial. Here, by applying an in vitro system enabling inducible gene expression control, we report that moderate induction of transcriptionally active exogenous ß-catenin in ß-catenin null mouse ESCs promotes epiblast-like cell (EpiLC) derivation in vitro. Instead, in wild-type cells, moderate chemical pre-activation of the Wnt/ß-catenin pathway promotes EpiLC in vitro derivation. Finally, we suggest that moderate ß-catenin levels in ß-catenin null mouse ESCs favor early stem cell commitment toward mesoderm if the exogenous protein is induced only in the "ground state" of pluripotency condition, or endoderm if the induction is maintained during the differentiation. Overall, our results confirm previous findings about the role of ß-catenin in pluripotency and differentiation, while indicating a role for its doses in promoting specific differentiation programs.

TFEB Regulates ATP7B Expression to Promote Platinum Chemoresistance in Human Ovarian Cancer Cells.

ATP7B is a hepato-specific Golgi-located ATPase, which plays a key role in the regulation of copper (Cu) homeostasis and signaling. In response to elevated Cu levels, ATP7B traffics from the Golgi to endo-lysosomal structures, where it sequesters excess copper and further promotes its excretion to the bile at the apical surface of hepatocytes. In addition to liver, high ATP7B expression has been reported in tumors with elevated resistance to platinum (Pt)-based chemotherapy. Chemoresistance to Pt drugs represents the current major obstacle for the treatment of large cohorts of cancer patients. Although the mechanisms underlying Pt-tolerance are still ambiguous, accumulating evidence suggests that lysosomal sequestration of Pt drugs by ion transporters (including ATP7B) might significantly contribute to drug resistance development. In this context, signaling mechanisms regulating the expression of transporters such as ATP7B are of great importance. Considering this notion, we investigated whether ATP7B expression in Pt-resistant cells might be driven by transcription factor EB (TFEB), a master regulator of lysosomal gene transcription. Using resistant ovarian cancer IGROV-CP20 cells, we found that TFEB directly binds to the predicted coordinated lysosomal expression and regulation (CLEAR) sites in the proximal promoter and first intron region of ATP7B upon Pt exposure. This binding accelerates transcription of luciferase reporters containing ATP7B CLEAR regions, while suppression of TFEB inhibits ATP7B expression and stimulates cisplatin toxicity in resistant cells. Thus, these data have uncovered a Pt-dependent transcriptional mechanism that contributes to cancer chemoresistance and might be further explored for therapeutic purposes.

A gene toolbox for monitoring autophagy transcription.

Autophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the "induction list" by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps.

Integrated Genomics Identifies miR-181/TFAM Pathway as a Critical Driver of Drug Resistance in Melanoma.

MicroRNAs (miRNAs) are attractive therapeutic targets and promising candidates as molecular biomarkers for various therapy-resistant tumors. However, the association between miRNAs and drug resistance in melanoma remains to be elucidated. We used an integrative genomic analysis to comprehensively study the miRNA expression profiles of drug-resistant melanoma patients and cell lines. MicroRNA-181a and -181b (miR181a/b) were identified as the most significantly down-regulated miRNAs in resistant melanoma patients and cell lines. Re-establishment of miR-181a/b expression reverses the resistance of melanoma cells to the BRAF inhibitor dabrafenib. Introduction of miR-181 mimics markedly decreases the expression of TFAM in A375 melanoma cells resistant to BRAF inhibitors. Furthermore, melanoma growth was inhibited in A375 and M14 resistant melanoma cells transfected with miR-181a/b mimics, while miR-181a/b depletion enhanced resistance in sensitive cell lines. Collectively, our study demonstrated that miR-181a/b could reverse the resistance to BRAF inhibitors in dabrafenib resistant melanoma cell lines. In addition, miR-181a and -181b are strongly down-regulated in tumor samples from patients before and after the development of resistance to targeted therapies. Finally, melanoma tissues with high miR-181a and -181b expression presented favorable outcomes in terms of Progression Free Survival, suggesting that miR-181 is a clinically relevant candidate for therapeutic development or biomarker-based therapy selection.

Up-regulation of miR-34b/c by JNK and FOXO3 protects from liver fibrosis.

α1-Antitrypsin (AAT) deficiency is a common genetic disease presenting with lung and liver diseases. AAT deficiency results from pathogenic variants in the SERPINA1 gene encoding AAT and the common mutant Z allele of SERPINA1 encodes for Z α1-antitrypsin (ATZ), a protein forming hepatotoxic polymers retained in the endoplasmic reticulum of hepatocytes. PiZ mice express the human ATZ and are a valuable model to investigate the human liver disease of AAT deficiency. In this study, we investigated differential expression of microRNAs (miRNAs) between PiZ and control mice and found that miR-34b/c was up-regulated and its levels correlated with intrahepatic ATZ. Furthermore, in PiZ mouse livers, we found that Forkhead Box O3 (FOXO3) driving microRNA-34b/c (miR-34b/c) expression was activated and miR-34b/c expression was dependent upon c-Jun N-terminal kinase (JNK) phosphorylation on Ser574 Deletion of miR-34b/c in PiZ mice resulted in early development of liver fibrosis and increased signaling of platelet-derived growth factor (PDGF), a target of miR-34b/c. Activation of FOXO3 and increased miR-34c were confirmed in livers of humans with AAT deficiency. In addition, JNK-activated FOXO3 and miR-34b/c up-regulation were detected in several mouse models of liver fibrosis. This study reveals a pathway involved in liver fibrosis and potentially implicated in both genetic and acquired causes of hepatic fibrosis.

Loss of Ciliary Gene Bbs8 Results in Physiological Defects in the Retinal Pigment Epithelium.

Primary cilia are sensory organelles vital for developmental and physiological processes. Their dysfunction causes a range of phenotypes including retinopathies. Although primary cilia have been described in the retinal pigment epithelium (RPE), little is known about their contribution to biological processes within this tissue. Ciliary proteins are increasingly being identified in non-ciliary locations and might carry out additional functions, disruption of which possibly contributes to pathology. The RPE is essential for maintaining photoreceptor cells and visual function. We demonstrate that upon loss of Bbs8, predominantly thought to be a ciliary gene, the RPE shows changes in gene and protein expression initially involved in signaling pathways and developmental processes, and at a later time point RPE homeostasis and function. Differentially regulated molecules affecting the cytoskeleton and cellular adhesion, led to defective cellular polarization and morphology associated with a possible epithelial-to-mesenchymal transition (EMT)-like phenotype. Our data highlights the benefit of combinatorial "omics" approaches with in vivo data for investigating the function of ciliopathy proteins. It also emphasizes the importance of ciliary proteins in the RPE and their contribution to visual disorders, which must be considered when designing treatment strategies for retinal degeneration.

GADD34 is a modulator of autophagy during starvation.

Cells respond to starvation by shutting down protein synthesis and by activating catabolic processes, including autophagy, to recycle nutrients. This two-pronged response is mediated by the integrated stress response (ISR) through phosphorylation of eIF2α, which represses protein translation, and by inhibition of mTORC1 signaling, which promotes autophagy also through a stress-responsive transcriptional program. Implementation of such a program, however, requires protein synthesis, thus conflicting with general repression of translation. How is this mismatch resolved? We found that the main regulator of the starvation-induced transcriptional program, TFEB, counteracts protein synthesis inhibition by directly activating expression of GADD34, a component of the protein phosphatase 1 complex that dephosphorylates eIF2α. We discovered that GADD34 plays an essential role in autophagy by tuning translation during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux. Hence, the TFEB-GADD34 axis integrates the mTORC1 and ISR pathways in response to starvation.

MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B.

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.

CHOP and c-JUN up-regulate the mutant Z α1-antitrypsin, exacerbating its aggregation and liver proteotoxicity.

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop-/- mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop-/- mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter-based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.