PKCδ deficiency inhibits fetal development and is associated with heart elastic fiber hyperplasia and lung inflammation in adult PKCδ knockout mice

Protein kinase C-delta (PKCδ) has a caspase-3 recognition sequence in its structure, suggesting its involvement in apoptosis. In addition, PKCδ was recently reported to function as an anti-cancer factor. The generation of a PKCδ knockout mouse model indicated that PKCδ plays a role in B cell homeostasis. However, the Pkcrd gene, which is regulated through complex transcription, produces multiple proteins via alternative splicing. Since gene mutations can result in the loss of function of molecular species required for each tissue, in the present study, conditional PKCδ knockout mice lacking PKCδI, II, IV, V, VI, and VII were generated to enable tissue-specific deletion of PKCδ using a suitable Cre mouse. We generated PKCδ-null mice that lacked whole-body expression of PKCδ. PKCδ+/- parental mice gave birth to only 3.4% PKCδ-/- offsprings that deviated significantly from the expected Mendelian ratio (χ2(2) = 101.7, P < 0.001). Examination of mice on embryonic day 11.5 (E11.5) showed the proportion of PKCδ-/- mice implanted in the uterus in accordance with Mendelian rules; however, approximately 70% of the fetuses did not survive at E11.5. PKCδ-/- mice that survived until adulthood showed enlarged spleens, with some having cardiac and pulmonary abnormalities. Our findings suggest that the lack of PKCδ may have harmful effects on fetal development, and heart and lung functions after birth. Furthermore, our study provides a reference for future studies on PKCδ deficient mice that would elucidate the effects of the multiple protein variants in mice and decipher the roles of PKCδ in various diseases.

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A Melanocortin-4 Receptor Agonist Induces Skin and Hair Pigmentation in Patients with Monogenic Mutations in the Leptin-Melanocortin Pathway

Skin Pharmacology and Physiology ◽

10.1159/000516282 ◽

2021 ◽

pp. 1-10

Author(s):

Varvara Kanti ◽

Lia Puder ◽

Irina Jahnke ◽

Philipp Maximilian Krabusch ◽

Jan Kottner ◽

...

Keyword(s):

Leptin Receptor ◽

Skin Pigmentation ◽

Gene Mutations ◽

Continuous Treatment ◽

Whole Body ◽

Hair Color ◽

Phase 2 ◽

Loss Of Function ◽

The Impact ◽

Over Time

Background and Objectives: Gene mutations within the leptin-melanocortin signaling pathway lead to severe early-onset obesity. Recently, a phase 2 trial evaluated new pharmacological treatment options with the MC4R agonist setmelanotide in patients with mutations in the genes encoding proopiomelanocortin (POMC) and leptin receptor (LEPR). During treatment with setmelanotide, changes in skin pigmentation were observed, probably due to off-target effects on the closely related melanocortin 1 receptor (MC1R). Here, we describe in detail the findings of dermatological examinations and measurements of skin pigmentation during this treatment over time and discuss the impact of these changes on patient safety. Methods: In an investigator-initiated, phase 2, open-label pilot study, 2 patients with loss-of-function POMC gene mutations and 3 patients with loss-of-function variants in LEPR were treated with the MC4R agonist setmelanotide. Dermatological examination, dermoscopy, whole body photographic documentation, and spectrophotometric measurements were performed at screening visit and approximately every 3 months during the course of the study. Results: We report the results of a maximum treatment duration of 46 months. Skin pigmentation increased in all treated patients, as confirmed by spectrophotometry. During continuous treatment, the current results indicate that elevated tanning intensity levels may stabilize over time. Lips and nevi also darkened. In red-haired study participants, hair color changed to brown after initiation of setmelanotide treatment. Discussion: Setmelanotide treatment leads to skin tanning and occasionally hair color darkening in both POMC- and LEPR-deficient patients. No malignant skin changes were observed in the patients of this study. However, the results highlight the importance of regular skin examinations before and during MC4R agonist treatment.

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Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

Blood Advances ◽

10.1182/bloodadvances.2019000391 ◽

2019 ◽

Vol 3 (15) ◽

pp. 2355-2367 ◽

Cited By ~ 7

Author(s):

Yueh-Chwen Hsu ◽

Tsung-Chih Chen ◽

Chien-Chin Lin ◽

Chang-Tsu Yuan ◽

Chia-Lang Hsu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Knockout Mice ◽

Neurodevelopmental Disorder ◽

Loss Of Function ◽

Self Renewal ◽

Hematopoietic Stem ◽

Knockout Mouse Model ◽

The Impact

Abstract Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing 2 plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson-Forssman-Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T-cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8 weeks of age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared with the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin–c-Kit+Sca-1+ cells in the marrow of young Phf6 knockout mice. Functional studies, including competitive repopulation unit and serial transplantation assays, revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations, including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of key gene expression in those pathways. In summary, our studies show the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

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SIRT6 regulates metabolic homeostasis in skeletal muscle through activation of AMPK

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00122.2017 ◽

2017 ◽

Vol 313 (4) ◽

pp. E493-E505 ◽

Cited By ~ 26

Author(s):

Xiaona Cui ◽

Lu Yao ◽

Xiaoying Yang ◽

Yong Gao ◽

Fude Fang ◽

...

Keyword(s):

Skeletal Muscle ◽

Whole Body ◽

Acid Oxidation ◽

Metabolic Homeostasis ◽

Lipid Uptake ◽

Loss Of Function ◽

Knockout Mouse Model ◽

Expression Of Genes ◽

Amp Activated Protein Kinase ◽

Body Energy

Because of the mass and functions in metabolism, skeletal muscle is one of the major organs regulating whole body metabolic homeostasis. SIRT6, a histone deacetylase, has been shown to regulate metabolism in liver and brain; however, its specific role in skeletal muscle is undetermined. In the present study we explored physiological function of SIRT6 in muscle. We generated a muscle-specific SIRT6 knockout mouse model. The mice with SIRT6 deficiency in muscle displayed impaired glucose homeostasis and insulin sensitivity, attenuated whole body energy expenditure, and weakened exercise performance. Mechanistically, deletion of SIRT6 in muscle decreased expression of genes involved in glucose and lipid uptake, fatty acid oxidation, and mitochondrial oxidative phosphorylation in muscle cells because of the reduced AMP-activated protein kinase (AMPK) activity. In contrast, overexpression of SIRT6 in C2C12 myotubes activates AMPK. Our results from both gain- and loss-of-function experiments identify SIRT6 as a physiological regulator of muscle mitochondrial function. These findings indicate that SIRT6 is a potential therapeutic target for treatment of type 2 diabetes mellitus.

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Pathogenic STX3 variants affecting the retinal and intestinal transcripts cause an early-onset severe retinal dystrophy in microvillus inclusion disease subjects

Human Genetics ◽

10.1007/s00439-021-02284-1 ◽

2021 ◽

Author(s):

Andreas R. Janecke ◽

Xiaoqin Liu ◽

Rüdiger Adam ◽

Sumanth Punuru ◽

Arne Viestenz ◽

...

Keyword(s):

Early Onset ◽

Single Nucleotide Polymorphism Array ◽

Outer Nuclear Layer ◽

Retinal Dystrophy ◽

Geographic Origin ◽

Retinal Disease ◽

Homozygosity Mapping ◽

Rod Photoreceptor ◽

Loss Of Function ◽

Knockout Mouse Model

AbstractBiallelic STX3 variants were previously reported in five individuals with the severe congenital enteropathy, microvillus inclusion disease (MVID). Here, we provide a significant extension of the phenotypic spectrum caused by STX3 variants. We report ten individuals of diverse geographic origin with biallelic STX3 loss-of-function variants, identified through exome sequencing, single-nucleotide polymorphism array-based homozygosity mapping, and international collaboration. The evaluated individuals all presented with MVID. Eight individuals also displayed early-onset severe retinal dystrophy, i.e., syndromic—intestinal and retinal—disease. These individuals harbored STX3 variants that affected both the retinal and intestinal STX3 transcripts, whereas STX3 variants affected only the intestinal transcript in individuals with solitary MVID. That STX3 is essential for retinal photoreceptor survival was confirmed by the creation of a rod photoreceptor-specific STX3 knockout mouse model which revealed a time-dependent reduction in the number of rod photoreceptors, thinning of the outer nuclear layer, and the eventual loss of both rod and cone photoreceptors. Together, our results provide a link between STX3 loss-of-function variants and a human retinal dystrophy. Depending on the genomic site of a human loss-of-function STX3 variant, it can cause MVID, the novel intestinal-retinal syndrome reported here or, hypothetically, an isolated retinal dystrophy.

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Minimal impact of age and housing temperature on the metabolic phenotype of Acc2−/− mice

Journal of Endocrinology ◽

10.1530/joe-15-0444 ◽

2015 ◽

Vol 228 (3) ◽

pp. 127-134 ◽

Cited By ~ 6

Author(s):

Amanda E Brandon ◽

Ella Stuart ◽

Simon J Leslie ◽

Kyle L Hoehn ◽

David E James ◽

...

Keyword(s):

Skeletal Muscle ◽

Body Composition ◽

Energy Expenditure ◽

Glucose Tolerance ◽

Fat Mass ◽

Knockout Mice ◽

Muscle Glycogen ◽

Insulin Action ◽

Metabolic Phenotype ◽

Whole Body

An important regulator of fatty acid oxidation (FAO) is the allosteric inhibition of CPT-1 by malonyl-CoA produced by the enzyme acetyl-CoA carboxylase 2 (ACC2). Initial studies suggested that deletion of Acc2 (Acacb) increased fat oxidation and reduced adipose tissue mass but in an independently generated strain of Acc2 knockout mice we observed increased whole-body and skeletal muscle FAO and a compensatory increase in muscle glycogen stores without changes in glucose tolerance, energy expenditure or fat mass in young mice (12–16 weeks). The aim of the present study was to determine whether there was any effect of age or housing at thermoneutrality (29 °C; which reduces total energy expenditure) on the phenotype of Acc2 knockout mice. At 42–54 weeks of age, male WT and Acc2−/− mice had similar body weight, fat mass, muscle triglyceride content and glucose tolerance. Consistent with younger Acc2−/− mice, aged Acc2−/− mice showed increased whole-body FAO (24 h average respiratory exchange ratio=0.95±0.02 and 0.92±0.02 for WT and Acc2−/− mice respectively, P<0.05) and skeletal muscle glycogen content (+60%, P<0.05) without any detectable change in whole-body energy expenditure. Hyperinsulinaemic–euglycaemic clamp studies revealed no difference in insulin action between groups with similar glucose infusion rates and tissue glucose uptake. Housing Acc2−/− mice at 29 °C did not alter body composition, glucose tolerance or the effects of fat feeding compared with WT mice. These results confirm that manipulation of Acc2 may alter FAO in mice, but this has little impact on body composition or insulin action.

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Functional characterization of a loss-of-function mutant I324M of arginine vasopressin receptor 2 in X-linked nephrogenic diabetes insipidus

Scientific Reports ◽

10.1038/s41598-021-90736-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lixia Wang ◽

Weihong Guo ◽

Chunyun Fang ◽

Wenli Feng ◽

Yumeng Huang ◽

...

Keyword(s):

Diabetes Insipidus ◽

Arginine Vasopressin ◽

Nephrogenic Diabetes Insipidus ◽

Functional Characterization ◽

Gene Mutations ◽

Biochemical Properties ◽

Vasopressin Receptor ◽

Inherited Disease ◽

Loss Of Function ◽

Gene Encoding

AbstractX-linked nephrogenic diabetes insipidus (X-linked NDI) is a rare inherited disease mainly caused by lost-of-function mutations in human AVPR2 gene encoding arginine vasopressin receptor 2 (V2R). Our focus of the current study is on exploration of the functional and biochemical properties of Ile324Met (I324M) mutation identified in a pedigree showing as typical recessive X-linked NDI. We demonstrated that I324M mutation interfered with the conformation of complex glycosylation of V2R. Moreover, almost all of the I324M-V2R failed to express on the cell surface due to being captured by the endoplasmic reticulum control system. We further examined the signaling activity of DDAVP-medicated cAMP and ERK1/2 pathways and the results revealed that the mutant receptor lost the ability in response to DDAVP stimulation contributed to the failure of accumulation of cAMP and phosphorylated ERK1/2. Based on the characteristics of molecular defects of I324M mutant, we selected two reagents (SR49059 and alvespimycin) to determine whether the functions of I324M-V2R can be restored and we found that both compounds can significantly “rescue” I324M mutation. Our findings may provide further insights for understanding the pathogenic mechanism of AVPR2 gene mutations and may offer some implications on development of promising treatments for patients with X-linked NDI.

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The transcription factor E2F-1 mediates the autoregulation of RB gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.299-309.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 299-309

Author(s):

B Shan ◽

C Y Chang ◽

D Jones ◽

W H Lee

Keyword(s):

Promoter Region ◽

Promoter Activity ◽

Cell Cycle Progression ◽

Gene Mutations ◽

Suppressor Gene ◽

Recognition Sequence ◽

Cycle Progression ◽

Rb Gene ◽

Transcription Factor E2f ◽

Stimulation Of

The retinoblastoma (RB) gene is the prototype tumor suppressor gene. Mutations in this gene are often associated with the occurrence of various tumors. Several mutations have been found in the promoter region of the gene, suggesting that inappropriate transcriptional regulation of the RB gene contributes to tumorigenesis. Sequence analysis of the RB promoter has revealed a potential E2F recognition site within a region critical for RB gene transcription. By using the cloned E2F-1 gene, here we report that (i) RB expression is negatively regulated by its own gene product, (ii) E2F-1 binds specifically to an E2F recognition sequence in the RB promoter and transactivates the RB promoter, (iii) overexpression of RB suppresses E2F-1-mediated stimulation of RB promoter activity, and (iv) the expression of the RB gene is paralleled by the expression of the E2F-1 gene during cell cycle progression. These results demonstrate that expression of RB is negatively autoregulated through E2F-1.

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The domino gene of Drosophila encodes novel members of the SWI2/SNF2 family of DNA-dependent ATPases, which contribute to the silencing of homeotic genes

Development ◽

10.1242/dev.128.8.1429 ◽

2001 ◽

Vol 128 (8) ◽

pp. 1429-1441 ◽

Cited By ~ 6

Author(s):

M.L. Ruhf ◽

A. Braun ◽

O. Papoulas ◽

J.W. Tamkun ◽

N. Randsholt ◽

...

Keyword(s):

Polytene Chromosomes ◽

Gene Mutations ◽

Polycomb Group ◽

Protein Isoforms ◽

Homeotic Genes ◽

Loss Of Function ◽

Trithorax Group ◽

A Subunit ◽

The Common ◽

Hematopoietic Disorders

The Drosophila domino gene has been isolated in a screen for mutations that cause hematopoietic disorders. Generation and analysis of loss-of-function domino alleles show that the phenotypes are typical for proliferation gene mutations. Clonal analysis demonstrates that domino is necessary for cell viability and proliferation, as well as for oogenesis. domino encodes two protein isoforms of 3202 and 2498 amino acids, which contain a common N-terminal region but divergent C termini. The common region includes a 500 amino acid DNA-dependent ATPase domain of the SWI2/SNF2 family of proteins, which function via interaction with chromatin. We show that, although domino alleles do not exhibit homeotic phenotypes by themselves, domino mutations enhance Polycomb group mutations and counteract Trithorax group effects. The Domino proteins are present in large complexes in embryo extracts, and one isoform binds to a number of discrete sites on larval polytene chromosomes. Altogether, the data lead us to propose that domino acts as a repressor by interfering with chromatin structure. This activity is likely to be performed as a subunit of a chromatin-remodeling complex.

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Abstract 15813: Loss-of-Function Mutation in Toll-Like Receptor 4 Partially Protects Against Peripheral and Cardiac Insulin Resistance During a Long-Term High-Fat Diet

Circulation ◽

10.1161/circ.130.suppl_2.15813 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Ellen Jackson ◽

Elizabeth Rendina-Ruedy ◽

Matt Priest ◽

Brenda Smith ◽

Veronique Lacombe

Keyword(s):

Insulin Resistance ◽

Protein Content ◽

Glucose Transport ◽

High Fat Diet ◽

Whole Body ◽

Toll Like Receptor ◽

Loss Of Function ◽

High Fat ◽

The Face ◽

Tlr4 Activation

Diabetes mellitus is an epidemic disease characterized by alterations in glucose transport, which is tightly regulated by a family of specialized proteins called the glucose transporters (GLUTs). Although diabetic cardiomyopathy is a common complication in diabetic patients, its pathogenesis is still not well understood. Toll-like receptor (TLR) 4, which plays a central role in pathogen recognition by the innate immune system, may also play a critical role in linking inflammation and metabolic disease. We hypothesized that TLR4 activation triggers cardiac insulin resistance. We used mice with a loss-of function mutation in TLR4 (C3H/HeJ) and age-matched wild-type (WT, C57BL/6N) mice (n=8/group) to investigate how feeding a high-fat diet (HFD, 60% kcal from fat) for 16 weeks affected whole-body and cardiac glucose metabolism. After 16 weeks, WT mice fed a HFD were obese and developed hyperglycemia and insulin resistance compared to WT mice on a control diet (10% kcal from fat). The C3H/HeJ mice were partially protected against HFD-induced obesity and insulin resistance. In the heart, WT mice fed a HFD had a 30% decrease (P<0.05) in GLUT4 protein content as measured by Western Blot of cardiac crude membrane protein extracts. In contrast, the loss-of-function point mutation in TLR4 partially rescued cardiac GLUT4 content in the face of a HFD. Interestingly, there was a 40% increase (P<0.05) in the novel GLUT isoform, GLUT8, in the heart when mice of either genotype were fed a HFD. Additionally, GLUT4 protein content was negatively (P<0.05) correlated with GLUT8 content in the myocardium, suggesting that GLUT8 may act as a compensatory mechanism in the face of HFD-induced GLUT4 downregulation. Phosphorylated Akt, a key protein of the insulin signaling pathway, was positively (P<0.05) correlated with GLUT4 content, while the basal/inactive form was negatively correlated. In conclusion, these data suggest that activation of TLR4 activation during diabetes and obesity alters glucose transport by an Akt mechanism, and as such is a pathogenic factor during peripheral and cardiac insulin resistance. Overall, TLR4 appears to be a key modulator in the cross-talk between inflammatory and metabolic pathways, as well as a potential therapeutic target for diabetes.

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PCNT point mutations and familial intracranial aneurysms

Neurology ◽

10.1212/wnl.0000000000006614 ◽

2018 ◽

Vol 91 (23) ◽

pp. e2170-e2181 ◽

Cited By ~ 6

Author(s):

Oswaldo Lorenzo-Betancor ◽

Patrick R. Blackburn ◽

Emily Edwards ◽

Rocío Vázquez-do-Campo ◽

Eric W. Klee ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Intracranial Aneurysms ◽

Point Mutations ◽

Missense Mutations ◽

Loss Of Function ◽

Interaction Domain ◽

Knockout Mouse Model ◽

Whole Exome ◽

Primordial Dwarfism

ObjectiveTo identify novel genes involved in the etiology of intracranial aneurysms (IAs) or subarachnoid hemorrhages (SAHs) using whole-exome sequencing.MethodsWe performed whole-exome sequencing in 13 individuals from 3 families with an autosomal dominant IA/SAH inheritance pattern to look for candidate genes for disease. In addition, we sequenced PCNT exon 38 in a further 161 idiopathic patients with IA/SAH to find additional carriers of potential pathogenic variants.ResultsWe identified 2 different variants in exon 38 from the PCNT gene shared between affected members from 2 different families with either IA or SAH (p.R2728C and p.V2811L). One hundred sixty-four samples with either SAH or IA were Sanger sequenced for the PCNT exon 38. Five additional missense mutations were identified. We also found a second p.V2811L carrier in a family with a history of neurovascular diseases.ConclusionThe PCNT gene encodes a protein that is involved in the process of microtubule nucleation and organization in interphase and mitosis. Biallelic loss-of-function mutations in PCNT cause a form of primordial dwarfism (microcephalic osteodysplastic primordial dwarfism type II), and ≈50% of these patients will develop neurovascular abnormalities, including IAs and SAHs. In addition, a complete Pcnt knockout mouse model (Pcnt−/−) published previously showed general vascular abnormalities, including intracranial hemorrhage. The variants in our families lie in the highly conserved PCNT protein-protein interaction domain, making PCNT a highly plausible candidate gene in cerebrovascular disease.

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