In eukaryotic cells, there are two well characterized pathways that regulate translation initiation in response to stress, and each have been shown to be targeted by various viruses. We recently ...showed in a yeast-based model that the bacterial virulence factor YopJ disrupts one of these pathways, which is centered on the α-subunit of the translation factor eIF2. Here, we show in mammalian cells that induction of the eIF2 signaling pathway occurs following infection with bacterial pathogens and that, consistent with our yeast-based findings, YopJ reduces eIF2 signaling in response to endoplasmic reticulum stress, heavy metal toxicity, dsRNA, and bacterial infection. We demonstrate that the well documented activities of YopJ, inhibition of NF-κB activation and proinflammatory cytokine expression, are both dependent on an intact eIF2 signaling pathway. Unexpectedly, we found that cells with defective eIF2 signaling were more susceptible to bacterial invasion. This was true for pathogenic Yersinia, a facultative intracellular pathogen, as well as for the intracellular pathogens Listeria monocytogenes and Chlamydia trachomatis. Collectively, our data indicate that the highly conserved eIF2 signaling pathway, which is vitally important for antiviral responses, plays a variety of heretofore unrecognized roles in antibacterial responses.
Background: eIF2 is a critical point of stress-induced regulation of translation in eukaryotic cells.
Results: eIF2 signaling is activated by bacterial pathogens and regulates two key infection-associated processes.
Conclusion: Regulation of translation in eukaryotic cells is involved in innate immune responses.
Significance: These findings enlarge the possible targets for therapeutic interventions against bacterial pathogens.
Fibrosis is a major cause of loss of renal function in autosomal dominant polycystic kidney disease (ADPKD). In this study, we examined whether vasopressin type-2 receptor (V2R) activity in cystic ...epithelial cells can stimulate interstitial myofibroblasts and fibrosis in ADPKD kidneys.
We treated
gene knockout (
KO) mice with dDAVP, a V2R agonist, for 3 days and evaluated the effect on myofibroblast deposition of extracellular matrix (ECM). We also analyzed the effects of conditioned media from primary cultures of human ADPKD cystic epithelial cells on myofibroblast activation. Because secretion of the profibrotic connective tissue growth factor (CCN2) increased significantly in dDAVP-treated
KO mouse kidneys, we examined its role in V2R-dependent fibrosis in ADPKD as well as that of yes-associated protein (YAP).
V2R stimulation using dDAVP increased the renal interstitial myofibroblast population and ECM deposition. Similarly, conditioned media from human ADPKD cystic epithelial cells increased myofibroblast activation
, suggesting a paracrine mechanism. Renal collecting duct-specific gene deletion of
significantly reduced cyst growth and myofibroblasts in
KO mouse kidneys. We found that YAP regulates
, and YAP inhibition or gene deletion reduces renal fibrosis in
KO mouse kidneys. Importantly, YAP inactivation blocks the dDAVP-induced increase in myofibroblasts in
KO kidneys. Further
studies showed that V2R regulates YAP by an ERK1/2-dependent mechanism in human ADPKD cystic epithelial cells.
Our results demonstrate a novel mechanism by which cystic epithelial cells stimulate myofibroblasts in the pericystic microenvironment, leading to fibrosis in ADPKD. The V2R-YAP-CCN2 cell signaling pathway may present a potential therapeutic target for fibrosis in ADPKD.
It has been appreciated for almost 20 years that members of the Chlamydiales possess a virulence-associated type III secretion mechanism. Given the obligate intracellular nature of these bacteria, ...defining exactly how type III secretion functions to promote pathogenesis has been challenging. We present a working model herein that is based on current evidence.
Dynamic interactions that govern the balance between host and pathogen determine the outcome of infection and are shaped by evolutionary pressures. Eukaryotic hosts have evolved elaborate and ...formidable defense mechanisms that provide the basis for innate and adaptive immunity. Proteins containing a membrane attack complex/Perforin (MACPF) domain represent an important class of immune effectors. These pore-forming proteins induce cell killing by targeting microbial or host membranes. Intracellular bacteria can be shielded from MACPF-mediated killing, and
spp. represent a successful paradigm of obligate intracellular parasitism. Ancestors of present-day
likely originated at evolutionary times that correlated with or preceded many host defense pathways. We discuss the current knowledge regarding how chlamydiae interact with the MACPF proteins Complement C9, Perforin-1, and Perforin-2. Current evidence indicates a degree of resistance by
to MACPF effector mechanisms. In fact, chlamydiae have acquired and adapted their own MACPF-domain protein to facilitate infection.
Lung squamous cell carcinoma (LSCC) is a prevalent form of lung cancer exhibiting distinctive histological and genetic characteristics. Chromosome 3q26 copy number gain (CNG) is a genetic hallmark of ...LSCC present in >90% of tumors. We report that 3q26 CNGs occur early in LSCC tumorigenesis, persist during tumor progression, and drive coordinate overexpression of PRKCI, SOX2, and ECT2. Overexpression of PRKCI, SOX2, and ECT2 in the context of Trp53 loss is sufficient to transform mouse lung basal stem cells into tumors with histological and genomic features of LSCC. Functionally, PRKCI and SOX2 collaborate to activate an extensive transcriptional program that enforces a lineage-restricted LSCC phenotype, whereas PRKCI and ECT2 collaborate to promote oncogenic growth. Gene signatures indicative of PKCι-SOX2 and PKCι-ECT2 signaling activity are enriched in the classical subtype of human LSCC and predict distinct therapeutic vulnerabilities. Thus, the PRKCI, SOX2, and ECT2 oncogenes represent a multigenic driver of LSCC.
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•3q26 CNG and TP53 mutation associate with progression of carcinoma in situ to LSCC•PRKCI, SOX2, and ECT2 overexpression transforms Trp53−/− mouse lung basal stem cells•Active PKCι-SOX2 and PKCι-ECT2 signaling is required for LBSC transformation•A PKCι-SOX2 molecular signature suggests unique therapeutic vulnerabilities in LSCC
Liu et al. report that three oncogenes, PRKCI, SOX2, and ECT2, which are coordinately amplified and overexpressed in lung squamous cell carcinoma (LSCC), can transform Trp53−/− mouse lung basal stem cells into tumors with histological and genomic features of LSCC and drive oncogenic signaling necessary to maintain a LSCC phenotype.
The human adaptive immune system must generate extraordinary diversity to be able to respond to all possible pathogens. The T-cell repertoire derives this high diversity through somatic recombination ...of the T-cell receptor (TCR) locus, a random process that results in repertoires that are largely private to each individual. However, factors such as thymic selection and T-cell proliferation upon antigen exposure can affect TCR sharing among individuals. By immunosequencing the TCRβ variable region of 426 healthy individuals, we find that, on average, fewer than 1% of TCRβ clones are shared between individuals, consistent with largely private TCRβ repertoires. However, we detect a significant correlation between increased HLA allele sharing and increased number of shared TCRβ clones, with each additional shared HLA allele contributing to an increase in ~0.01% of the total shared TCRβ clones, supporting a key role for HLA type in shaping the immune repertoire. Surprisingly, we find that shared antigen exposure to CMV leads to fewer shared TCRβ clones, even after controlling for HLA, indicative of a largely private response to major viral antigenic exposure. Consistent with this hypothesis, we find that increased age is correlated with decreased overall TCRβ clone sharing, indicating that the pattern of private TCRβ clonal expansion is a general feature of the T-cell response to other infectious antigens as well. However, increased age also correlates with increased sharing among the lowest frequency clones, consistent with decreased repertoire diversity in older individuals. Together, all of these factors contribute to shaping the TCRβ repertoire, and understanding their interplay has important implications for the use of T cells for therapeutics and diagnostics.
The giant sarcomere protein titin is important in both heart health and disease. Mutations in the gene encoding for titin (
) are the leading known cause of familial dilated cardiomyopathy. The ...uneven distribution of these mutations within
motivated us to seek a more complete understanding of this gene and the isoforms it encodes in cardiomyocyte (CM) sarcomere formation and function.
To investigate the function of titin in human CMs, we used CRISPR/Cas9 to generate homozygous truncations in the Z disk (TTN-Z
) and A-band (TTN-A
) regions of the
gene in human induced pluripotent stem cells. The resulting CMs were characterized with immunostaining, engineered heart tissue mechanical measurements, and single-cell force and calcium measurements.
After differentiation, we were surprised to find that despite the more upstream mutation, TTN-Z
-CMs had sarcomeres and visibly contracted, whereas TTN-A
-CMs did not. We hypothesized that sarcomere formation was caused by the expression of a recently discovered isoform of titin, Cronos, which initiates downstream of the truncation in TTN-Z
-CMs. Using a custom Cronos antibody, we demonstrate that this isoform is expressed and integrated into myofibrils in human CMs. TTN-Z
-CMs exclusively express Cronos titin, but these cells produce lower contractile force and have perturbed myofibril bundling compared with controls expressing both full-length and Cronos titin. Cronos titin is highly expressed in human fetal cardiac tissue, and when knocked out in human induced pluripotent stem cell derived CMs, these cells exhibit reduced contractile force and myofibrillar disarray despite the presence of full-length titin.
We demonstrate that Cronos titin is expressed in developing human CMs and is able to support partial sarcomere formation in the absence of full-length titin. Furthermore, Cronos titin is necessary for proper sarcomere function in human induced pluripotent stem cell derived CMs. Additional investigation is necessary to understand the molecular mechanisms of this novel isoform and how it contributes to human cardiac disease.
Histone methylation is an important regulator of gene expression; its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of telomere silencing 1-like ...(DOT1L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot1L-mutant mice to determine influence of this enzyme on embryonic hematopoiesis. Mutant mice developed more slowly than wild-type embryos and died between embryonic days 10.5 and 13.5, displaying a striking anemia, especially apparent in small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid, differentiation was observed. Erythroid progenitors failed to develop normally, showing retarded progression through the cell cycle, accumulation during G0/G1 stage, and marked increase in apoptosis in response to erythroid growth factors. GATA2, a factor essential for early erythropoiesis, was significantly reduced in Dot1L-deficient cells, whereas expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT1L-dependent lysine 79 of histone H3 methylation serves as a critical regulator of a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription, thus controlling numbers of circulating erythroid and myeloid cells.
End plates serve as the interface between rigid vertebral bodies and pliant intervertebral disks. Because the lumbar spine carries significant forces and disks don't have a dedicated blood supply, ...end plates must balance conflicting requirements of being strong to prevent vertebral fracture and porous to facilitate transport between disk cells and vertebral capillaries. Consequently, end plates are particularly susceptible to damage, which can increase communication between proinflammatory disk constituents and vascularized vertebral bone marrow. Damaged end plate regions can be sites of reactive bone marrow lesions that include proliferating nerves, which are susceptible to chemical sensitization and mechanical stimulation. Although several lines of evidence indicate that innervated end plate damage can be a source of chronic low back pain, its role in patients is likely underappreciated because innervated damage is poorly visualized with diagnostic imaging. This literature review summarizes end plate biophysical function and aspects of pathologic degeneration that can lead to vertebrogenic pain. Areas of future research are identified in the context of unmet clinical needs for patients with chronic low back pain.
Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector ...molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.
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