Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using ...quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or "spreading" of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.
Caveolae are the primary route for internalization and transendothelial transport of macromolecules, such as insulin and albumin. Caveolae-mediated endocytosis is activated by Src-dependent ...caveolin-1 (Cav-1) phosphorylation and subsequent recruitment of dynamin-2 and filamin A (FilA), which facilitate vesicle fission and trafficking, respectively. Here, we tested the role of RalA and phospholipase D (PLD) signaling in the regulation of caveolae-mediated endocytosis and trafficking. The addition of albumin to human lung microvascular endothelial cells induced the activation of RalA within minutes, and siRNA-mediated down-regulation of RalA abolished fluorescent BSA uptake. Co-immunoprecipitation studies revealed that albumin induced the association between RalA, Cav-1, and FilA; however, RalA knockdown with siRNA did not affect FilA recruitment to Cav-1, suggesting that RalA was not required for FilA and Cav-1 complex formation. Rather, RalA probably facilitates caveolae-mediated endocytosis by activating downstream effectors. PLD2 was shown to be activated by RalA, and inhibition of PLD2 abolished Alexa-488-BSA uptake, indicating that phosphatidic acid (PA) generated by PLD2 may facilitate caveolae-mediated endocytosis. Furthermore, using a PA biosensor, GFP-PASS, we observed that BSA induced an increase in PA co-localization with Cav-1-RFP, which could be blocked by a dominant negative PLD2 mutant. Total internal reflection fluorescence microscopy studies of Cav-1-RFP also showed that fusion of caveolae with the basal plasma membrane was dependent on PLD2 activity. Thus, our results suggest that the small GTPase RalA plays an important role in promoting invagination and trafficking of caveolae, not by potentiating the association between Cav-1 and FilA but by stimulating PLD2-mediated generation of phosphatidic acid.
SELENOF is a member of the class of selenoproteins in which the amino acid selenocysteine is co-translationally inserted into the elongating peptide in response to an in-frame UGA codon located in ...the 3'-untranslated (3'-UTR) region of the SELENOF mRNA. Polymorphisms in the 3'-UTR are associated with an increased risk of dying from prostate cancer and these variations are functional and 10 times more frequent in the genomes of African American men. SELENOF is dramatically reduced in prostate cancer compared to benign adjacent regions. Using a prostate cancer tissue microarray, it was previously established that the reduction of SELENOF in the cancers from African American men was significantly greater than in cancers from Caucasian men. When SELENOF levels in human prostate immortalized epithelial cells were reduced with an shRNA construct, those cells acquired the ability to grow in soft agar, increased the ability to migrate in a scratch assay and acquired features of energy metabolism associated with prostate cancer. These results support a role of SELENOF loss in prostate cancer progression and further indicate that SELENOF loss and genotype may contribute to the disparity in prostate cancer mortality experienced by African American men.
During cerebral cortical development, neural precursor-precursor interactions in the ventricular zone neurogenic niche coordinate signaling pathways that regulate proliferation and differentiation. ...Previous studies with shRNA knockdown approaches indicated that N-cadherin adhesion between cortical precursors regulates β-catenin signaling, but the underlying mechanisms remained poorly understood.
Here, with conditional knockout approaches, we find further supporting evidence that N-cadherin maintains β-catenin signaling during cortical development. Using shRNA to N-cadherin and dominant negative N-cadherin overexpression in cell culture, we find that N-cadherin regulates Wnt-stimulated β-catenin signaling in a cell-autonomous fashion. Knockdown or inhibition of N-cadherin with function-blocking antibodies leads to reduced activation of the Wnt co-receptor LRP6. We also find that N-cadherin regulates β-catenin via AKT, as reduction of N-cadherin causes decreased AKT activation and reduced phosphorylation of AKT targets GSK3β and β-catenin. Inhibition of AKT signaling in neural precursors in vivo leads to reduced β-catenin-dependent transcriptional activation, increased migration from the ventricular zone, premature neuronal differentiation, and increased apoptotic cell death.
These results show that N-cadherin regulates β-catenin signaling through both Wnt and AKT, and suggest a previously unrecognized role for AKT in neuronal differentiation and cell survival during cortical development.
We determined differential gene expression in response to high glucose in lymphoblastoid cell lines derived from matched individuals with type 1 diabetes with and without retinopathy. Those genes ...exhibiting the largest difference in glucose response were assessed for association with diabetic retinopathy in a genome-wide association study meta-analysis. Expression quantitative trait loci (eQTLs) of the glucose response genes were tested for association with diabetic retinopathy. We detected an enrichment of the eQTLs from the glucose response genes among small association p-values and identified folliculin (
) as a susceptibility gene for diabetic retinopathy. Expression of
in response to glucose was greater in individuals with diabetic retinopathy. Independent cohorts of individuals with diabetes revealed an association of
eQTLs with diabetic retinopathy. Mendelian randomization confirmed a direct positive effect of increased
expression on retinopathy. Integrating genetic association with gene expression implicated
as a disease gene for diabetic retinopathy.
Inflammatory epidermolysis bullosa acquisita (EBA) is characterized by a neutrophilic response to anti-type VII collagen (COL7) antibodies resulting in the development of skin inflammation and ...blistering. The antibody transfer model of EBA closely mirrors this EBA phenotype.
To better understand the changes induced in neutrophils upon recruitment from peripheral blood into lesional skin in EBA, we performed single-cell RNA-sequencing of whole blood and skin dissociate to capture minimally perturbed neutrophils and characterize their transcriptome.
Through this approach, we identified clear distinctions between circulating activated neutrophils and intradermal neutrophils. Most strikingly, the gene expression of multiple C-type lectin receptors, which have previously been reported to orchestrate host defense against fungi and select bacteria, were markedly dysregulated. After confirming the upregulation of
,
, and
in experimental EBA as well as in lesional skin from patients with inflammatory EBA, we performed functional studies in globally deficient
and
mice as well as in neutrophil-specific
mice. Deficiency in these genes did not reduce disease in the EBA model.
Collectively, our results suggest that while the upregulation of
,
, and
is a hallmark of activated dermal neutrophil populations, their individual contribution to the pathogenesis of EBA is dispensable.
Lung cancer is the leading cause of cancer death in the U.S. Therefore, it is imperative to identify novel biomarkers for the early detection and progression of lung cancer. PRMT6 is associated with ...poor lung cancer prognosis. However, analyzing PRMT6 expression manually in large samples is time-consuming posing a significant limitation for processing this biomarker. To overcome this issue, we trained and validated an automated method for scoring PRMT6 in lung cancer tissues, which can then be used as the standard method in future larger cohorts to explore population-level associations between PRMT6 expression and sociodemographic/clinicopathologic characteristics. We evaluated the ability of a trained artificial intelligence (AI) algorithm to reproduce the PRMT6 immunoreactive scores obtained by pathologists. Our findings showed that tissue segmentation to cancer vs. non-cancer tissues was the most critical parameter, which required training and adjustment of the algorithm to prevent scoring non-cancer tissues or ignoring relevant cancer cells. The trained algorithm showed a high concordance with pathologists with a correlation coefficient of 0.88. The inter-rater agreement was significant, with an intraclass correlation of 0.95 and a scale reliability coefficient of 0.96. In conclusion, we successfully optimized a machine learning algorithm for scoring PRMT6 expression in lung cancer that matches the degree of accuracy of scoring by pathologists.
A major limitation of current prostate cancer pharmacotherapy approaches is the inability of these compounds to target androgen receptor variants or mutants that develop during prostate cancer ...progression. The demand for novel therapeutics to prevent, slow, and treat prostate cancer is significant because FDA approved anti-androgens are associated with adverse events and can eventually drive drug-resistant prostate cancer. This study evaluated α-mangostin for its novel ability to degrade the androgen receptor and androgen receptor variants. α-Mangostin is one of more than 70 isoprenylated xanthones isolated from
that we have been evaluating for their anticancer potential. Prostate cancer cells treated with α-mangostin exhibited decreased levels of wild-type and mutated androgen receptors. Immunoblot, immunoprecipitation, and transfection experiments demonstrated that the androgen receptor was ubiquitinated and subsequently degraded via the proteasome, which we hypothesize occurs with the assistance of BiP, an ER chaperone protein that we have shown to associate with the androgen receptor. We also evaluated α-mangostin for its antitumor activity and promotion of androgen receptor degradation in vivo. In summary, our study demonstrates that androgen receptor degradation occurs through the novel activation of BiP and suggests a new therapeutic approach for prostate cancer.
Vesicle formation provides a means of cellular entry for extracellular substances and for recycling of membrane constituents. Mechanisms governing the two primary endocytic pathways (i.e., caveolae- ...and clathrin-mediated endocytosis, as well as newly emerging vesicular pathways) have become the focus of intense investigation to improve our understanding of nutrient, hormone, and drug delivery, as well as opportunistic invasion of pathogens. In this review of endocytosis, we broadly discuss the structural and signaling proteins that compose the molecular machinery governing endocytic vesicle formation (budding, invagination, and fission from the membrane), with some regard for the specificity observed in certain cell types and species. Important biochemical functions of endocytosis and diseases caused by their disruption also are discussed, along with the structures of key components of endocytic pathways and their known mechanistic contributions. The mechanisms by which principal components of the endocytic machinery are recruited to the plasma membrane, where they interact to induce vesicle formation, are discussed, together with computational approaches used to simulate simplified versions of endocytosis with the hope of clarifying aspects of vesicle formation that may be difficult to determine experimentally. Finally, we pose several unanswered questions intended to stimulate further research interest in the cell biology and modeling of endocytosis.
Chronic, nonhealing skin wounds, such as diabetic foot ulcers (DFUs), are common in patients with type 2 diabetes. Here, we investigated the role of chemokine (C-C motif) ligand 28 (CCL28) and its ...receptor C-C chemokine receptor type 10 (CCR10) in downregulation of endothelial nitric (NO) oxide synthase (eNOS) in association with delayed skin wound healing in the db/db mouse model of type 2 diabetes. We observed reduced eNOS expression and elevated CCL28/CCR10 levels in dorsal skin of db/db mice and subdermal leg biopsy specimens from human subjects with type 2 diabetes. Further interrogation revealed that overexpression of CCR10 reduced eNOS expression, NO bioavailability, and tube formation of human dermal microvascular endothelial cells (HDMVECs) in vitro, which was recapitulated in mouse dorsal skin. In addition, incubation of HDMVECs with CCL28 led to internalization of the CCR10/eNOS complex and colocalization with lysosome-associated membrane protein 1. Finally, topical application of myristoylated CCR10 binding domain 7 amino acid (Myr-CBD7) peptide prevented CCR10-eNOS interaction and subsequent eNOS downregulation, enhanced eNOS/NO levels, eNOS/VEGF-R2+ microvessel density, and blood perfusion, reduced inflammatory cytokine levels, and importantly, decreased wound healing time in db/db mice. Thus, endothelial cell CCR10 activation in genetically obese mice with type 2 diabetes promotes eNOS depletion and endothelial dysfunction, and targeted disruption of CCR10/eNOS interaction improves wound healing.
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