Jean Ann Maguire

Endoplasmic Reticulum Stress Induced by Surfactant Protein C BRICHOS Mutants Promotes Proinflammatory Signaling by Epithelial Cells

Chronic interstitial lung disease in both adults and children is associated with mutations of the surfactant protein C (SP-C) proprotein. Among these, mutations within the distal COOH propeptide, known as the BRICHOS domain, are associated with a severe disease phenotype. We showed that prolonged expression of the BRICHOS mutants, SP-C(Δexon4) and SP-C(L188Q), destabilizes endoplasmic reticulum (ER) quality-control mechanisms (the unfolded protein response, or UPR), resulting in the induction of ER stress signaling, an inhibition of the ubiquitin/proteasome system, and the activation of apoptotic pathways. Based on recent observations that the UPR and ER stress can be linked to the induction of proinflammatory signaling, we hypothesized that the epithelial cell dysfunction mediated by SP-C BRICHOS mutants would activate proinflammatory signaling pathways. In a test of this hypothesis, A549 and human embryonic kidney epithelial (HEK293) cells, transiently transfected with either SP-C(Δexon4) or SP-C(L188Q) mutants, each promoted the upregulation of multiple UPR response genes, including homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) and GRP78. Commensurate with these results, increases in IL-8 secretion occurred and were accompanied by the activation of c-Jun N-terminal kinase (JNK)/activating protein-1 signaling. The stimulation of IL-8 cytokine release was completely attenuated by treatment with the JNK-specific inhibitor, SP600125. In addition, SP-C(Δexon4), but not SP-C(L188Q), activated NFκB. The treatment of SP-C(Δexon4) transfected cells with 4-phenylbutyric acid, a small molecule chaperone known to improve protein folding, blocked the activation of NFκB, but not the release of IL-8. Taken together, the results support the role of JNK signaling in mediating SP-C BRICHOS-induced cytokine release, and provide a link between SP-C BRICHOS mutants and proinflammatory cytokine signaling.

Multiple ways to die: delineation of the unfolded protein response and apoptosis induced by Surfactant Protein C BRICHOS mutants.

Epithelial cell dysfunction is now recognized as an important mechanism in the pathogenesis of interstitial lung diseases. Surfactant Protein C (SP-C), an alveolar type II cell specific protein, has contributed to this concept with the observation that heterozygous expression of SFTPC gene mutations are associated with chronic interstitial lung disease. We have shown that transient expression of aggregation prone mutant SP-C isoforms (SP-C BRICHOS) destabilize ER quality control mechanisms resulting in the intracellular accumulation of aggregating propeptide, inhibition of the ubiquitin/proteasome system, and activation of apoptosis. The goal of the present study was to define signaling pathways linking the unfolded protein response (UPR) and subsequent ER stress with intrinsic apoptosis events observed following mutant SP-C expression. In vitro expression of the SP-C BRICHOS mutant, SP-C(Δexon4), was used as a model system. Here we show stimulation of a broad ER stress response in both transfected A549 and HEK293 cells with activation of all 3 canonical sensing pathways, IRE1/XBP-1, ATF6, and PERK/eIF2α. SP-C(Δexon4) expression also resulted in activation of caspase 3, but failed to stimulate expression of the apoptosis mediating transcription factors ATF4/CHOP. However, inhibition of either caspase 4 or c-jun kinase (JNK) each blocked caspase 3 mediated cell death. Taken together, these results suggest that expression of SP-C BRICHOS mutants induce apoptosis through multiple UPR signaling pathways, and provide new therapeutic targets for the amelioration of ER stress induced cytotoxicity observed in fibrotic lung remodeling.

A Nonaggregating Surfactant Protein C Mutant Is Misdirected to Early Endosomes and Disrupts Phospholipid Recycling

Interstitial lung disease in both children and adults has been linked to mutations in the lung‐specific surfactant protein C (SFTPC) gene. Among these, the missense mutation [isoleucine to threonine at codon 73 = human surfactant protein C (hSP‐CI73T)] accounts for ∼30% of all described SFTPC mutations. We reported previously that unlike the BRICHOS misfolding SFTPC mutants, expression of hSP‐CI73T induces lung remodeling and alveolar lipoproteinosis without a substantial Endoplasmic Reticulum (ER) stress response or ER‐mediated intrinsic apoptosis. We show here that, in contrast to its wild‐type counterpart that is directly routed to lysosomal‐like organelles for processing, SP‐CI73T is misdirected to the plasma membrane and subsequently internalized to the endocytic pathway via early endosomes, leading to the accumulation of abnormally processed proSP‐C isoforms. Functionally, cells expressing hSP‐CI73T demonstrated both impaired uptake and degradation of surfactant phospholipid, thus providing a molecular mechanism for the observed lipid accumulation in patients expressing hSP‐CI73T through the disruption of normal phospholipid recycling. Our data provide evidence for a novel cellular mechanism for conformational protein‐associated diseases and suggest a paradigm for mistargeted proteins involved in the disruption of the endosomal/lysosomal sorting machinery.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

GATA6 Plays an Important Role in the Induction of Human Definitive Endoderm, Development of the Pancreas, and Functionality of Pancreatic β Cells

Summary Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into β-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient’s disease. GATA6 mutant β-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and β-like cell functionality in humans.

Generation of human control iPS cell line CHOPWT9 from healthy adult peripheral blood mononuclear cells

The CHOPWT9 induced pluripotent stem (iPS) cell line was generated for use as a control for applications such as differentiation analyses to the three germ layers and derivative tissues. Peripheral blood mononuclear cells (PBMCs) obtained from a healthy adult female were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

Generation of Hermansky–Pudlak Syndrome Type 1 (HPS1) induced pluripotent stem cells (iPSCs)

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by deficiencies in lysosome-related organelles such as melanosomes and platelet-dense granules. The disorder is classified into nine different subtypes (HPS1-HPS9) based on genetic mutations in 9 unique genes. Here we describe the generation of an HPS1 iPSC line (CHOPHPS1) using a Cre-excisable polycistronic STEMCCA lentivirus. This line was derived from human fibroblasts isolated from a patient carrying a duplicative mutation in the HPS1 gene. The patient presented with oculocutaneous albinism, early pulmonary fibrosis, and hemorrhagic diathesis.

Generation of a human Juvenile myelomonocytic leukemia iPSC line, CHOPi001-A, with a mutation in CBL

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disorder of early childhood characterized by expansion of clonal myelomonocytic cells and hyperactive Ras/MAPK signaling. The disorder is caused by somatic and/or germline mutations in genes involved in the Ras/MAPK and JAK/STAT signaling pathways, including CBL. Here we describe the generation of an iPSC line with a homozygous CBL c.1111T->C (Y371H) mutation, designated CHOPJMML1854.

GATA6 suppression enhances lung specification from human pluripotent stem cells

&NA; The transcription factor GATA6 has been shown to be important for lung development and branching morphogenesis in mouse models, but its role in human lung development is largely unknown. Here, we studied the role of GATA6 during lung differentiation using human pluripotent stem cells. We found that the human stem cell lines most efficient at generating NKX2.1+ lung progenitors express lower endogenous levels of GATA6 during endoderm patterning and that knockdown of GATA6 during endoderm patterning increased the generation of these cells. Complete ablation of GATA6 resulted in the generation of lung progenitors displaying increased cell proliferation with up to a 15‐fold expansion compared with control cells, whereas the null cell line displayed a defect in further development into mature lung cell types. Furthermore, transgenic expression of GATA6 at the endoderm anteriorization stage skewed development toward a liver fate at the expense of lung progenitors. Our results suggest a critical dosage effect of GATA6 during human endoderm patterning and a later requirement during terminal lung differentiation. These studies offer an approach of modulating GATA6 expression to enhance the production of lung progenitors from human stem cell sources.

Highly Efficient CRISPR‐Cas9‐Mediated Genome Editing in Human Pluripotent Stem Cells

Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells.