Aladin M. Boriek

Nuclear factor-κB: its role in health and disease

Nuclear factor-κB (NF-κB) is a major transcription factor that plays an essential role in several aspects of human health including the development of innate and adaptive immunity. The dysregulation of NF-κB is associated with many disease states such as AIDS, atherosclerosis, asthma, arthritis, cancer, diabetes, inflammatory bowel disease, muscular dystrophy, stroke, and viral infections. Recent evidence also suggests that the dysfunction of NF-κB is a major mediator of some human genetic disorders. Appropriate regulation and control of NF-κB activity, which can be achieved by gene modification or pharmacological strategies, would provide a potential approach for the management of NF-κB related human diseases. This review summarizes the current knowledge of the physiological and pathophysiological functions of NF-κB and its possible role as a target of therapeutic intervention

Mechanical stress activates the nuclear factor-kappaB pathway in skeletal muscle fibers: a possible role in Duchenne muscular dystrophy

The ex vivo effects of passive mechanical stretch on the activation of nuclear factor‐kappaB (NF‐κB) pathways in skeletal muscles from normal and mdx mouse, a model of Duchenne muscular dystrophy (DMD), were investigated. The NF‐κB/DNA binding activity of the diaphragm muscle was increased by the application of axial mechanical stretch in a time‐dependent manner. The increased activation of NF‐κB was associated with a concomitant increase in I‐kappaB (IκB) kinase activity and the degradation of IκBμ protein. Pretreatment of the muscles with nifedipine (a Ca2+ channel blocker) and gadolinium(III) chloride (a stretch‐activated channel blocker) did not alter the level of activation of NF‐κB, ruling out involvement of Ca2+ influx through these channels. Furthermore, N‐acetyl cysteine, a free radical inhibitor, blocked the mechanical stretch‐induced NF‐κB activation, suggesting the involvement of free radicals. Compared with normal diaphragm, the basal level of NF‐κB activity was higher in muscles from mdx mice, and it was further enhanced in mechanically stretched muscles. Furthermore, activation of NF‐κB and increased expression of inflammatory cytokines IL‐1 β and tumor necrosis factor β in the mdx mouse precede the onset of muscular dystrophy. Our results show that mechanical stretch activates the classical NF‐κB pathway and this pathway could be predominately active in DMD. FASEB J. 17, 386–396 (2003)

Mechanical Stretch Up-regulates MicroRNA-26a and Induces Human Airway Smooth Muscle Hypertrophy by Suppressing Glycogen Synthase Kinase-3β

Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3′-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy.

Loss of dystrophin causes aberrant mechanotransduction in skeletal muscle fibers

Dystrophin is a cytoskeletal protein found at the inner surface of skeletal and cardiac muscle fibers. We hypothesize that deficiency of dystrophin increases muscle compliance and causes an aberrant mechanotransduction in muscle fibers. To test this hypothesis, we measured the length–tension relationships and determined intracellular signaling leading to the activation of mitogen‐activated protein (MAP) kinases in diaphragm muscle fibers from dystrophin‐deficient mdx mice. Compared with controls, length–tension curves of the mdx mice were shifted to the right. A higher level of activation of extracellular signal‐regulated kinase 1/2 (ERK1/2) but not c‐Jun N‐terminal kinase‐1 or p38 MAP kinase was observed in the mdx muscle compared with the normal muscle in response to mechanical stretch. Removal of Ca2+ from the medium inhibited stretch‐induced ERK1/2 activation only in mdx muscle fibers but not in the normal fibers. Conversely, pretreatment with TMB‐8 (an antagonist of intracellular Ca2+) blocked the mechanical stretchinduced ERK1/2 activation in normal but not in mdx muscle fibers. Pretreatment of muscle with nifedipine (L‐type calcium channel antagonist) marginally decreased the activation of ERK1/2 in normal or mdx muscle whereas pretreatment with gadolinium (III) chloride (an inhibitor of stretch‐activated channels) only blocked the activation of ERK1/2 in mdx muscle, with no significant effect on normal muscle. A higher basal level of activation of activator protein‐1 (AP‐1) transcription factor was observed in dystrophin‐deficient diaphragm, which was further augmented by mechanical stretch. Mechanical stretch‐induced activation of AP‐1 was decreased by pretreatment of muscle fibers with PD98059 (ERK1/2 inhibitor) and removal of Ca2+ ions from incubation medium. Our results show that dystrophin is a load‐bearing element and its deficiency leads to loss of muscle stiffness and aberrant mechanotransduction in skeletal muscle fibers.—Kumar, A., Khandelwal, N., Malya, R., Reid, M. B., Boriek, A. M. Loss of dystrophin causes aberrant mechanotransduction in skeletal muscle fibers. FASEB J. 18, 102–113 (2004)

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Mechanical stretch activates nuclear factor-kappaB, activator protein-1, and mitogen-activated protein kinases in lung parenchyma: implications in asthma

We investigated the effects of mechanical stretch and induced stimulation of lung parenchyma on the activation of proinflammatory transcription factors in normal mice and in a mouse model of asthma. Mechanical stretching of lung parenchyma led to increased activation of NF‐κB and AP‐1 transcription factors. Incubation of lung parenchyma with methacholine increased the activation of NF‐κB, which was further augmented by stretch. Activation of NF‐κB in response to mechanical stretch was associated with the phosphorylation and degradation of IκBα and the activation of IκB kinase. Stretch‐induced activation of NF‐κB involves activation of stretch‐activated (SA) channels and the production of free radicals. Mechanical stretch and/or treatment with methacholine resulted in an increased activation of ERK1/2 and p38 MAP kinase, and the inhibition of the activity of these kinases partially blocked the stretch‐induced NF‐κB and AP‐1 activation. A greater level of NF‐κB and ERK1/2 activity was observed in the asthmatic mice, which was further increased by mechanical stretching. The level of cyclooxygenase‐2, an NF‐κB‐regulated enzyme, was also higher in lung parenchyma from asthmatic mice than in normal mice. Our data suggest that mechanical stretching of lung parenchyma activates NF‐κB and AP‐1, at least in part, through the activation of MAP kinase signaling pathways.—Kumar, A., Lnu, S., Malya, R., Barron, D., Moore, J., Corry, D. B., Boriek, A. M. Mechanical stretch activates nuclear factor‐kappaB, activator protein‐1, and mitogen‐activated protein kinases in lung parenchyma: implications in asthma. FASEB J. 17, 1800–1811 (2003)

Association between Functional Small Airway Disease and FEV1 Decline in Chronic Obstructive Pulmonary Disease.

RATIONALE The small conducting airways are the major site of airflow obstruction in chronic obstructive pulmonary disease and may precede emphysema development. OBJECTIVES We hypothesized a novel computed tomography (CT) biomarker of small airway disease predicts FEV1 decline. METHODS We analyzed 1,508 current and former smokers from COPDGene with linear regression to assess predictors of change in FEV1 (ml/yr) over 5 years. Separate models for subjects without and with airflow obstruction were generated using baseline clinical and physiologic predictors in addition to two novel CT metrics created by parametric response mapping (PRM), a technique pairing inspiratory and expiratory CT images to define emphysema (PRM(emph)) and functional small airways disease (PRM(fSAD)), a measure of nonemphysematous air trapping. MEASUREMENTS AND MAIN RESULTS Mean (SD) rate of FEV1 decline in ml/yr for GOLD (Global Initiative for Chronic Obstructive Lung Disease) 0-4 was as follows: 41.8 (47.7), 53.8 (57.1), 45.6 (61.1), 31.6 (43.6), and 5.1 (35.8), respectively (trend test for grades 1-4; P < 0.001). In multivariable linear regression, for participants without airflow obstruction, PRM(fSAD) but not PRM(emph) was associated with FEV1 decline (P < 0.001). In GOLD 1-4 participants, both PRM(fSAD) and PRM(emph) were associated with FEV1 decline (P < 0.001 and P = 0.001, respectively). Based on the model, the proportional contribution of the two CT metrics to FEV1 decline, relative to each other, was 87% versus 13% and 68% versus 32% for PRM(fSAD) and PRM(emph) in GOLD 1/2 and 3/4, respectively. CONCLUSIONS CT-assessed functional small airway disease and emphysema are associated with FEV1 decline, but the association with functional small airway disease has greatest importance in mild-to-moderate stage chronic obstructive pulmonary disease where the rate of FEV1 decline is the greatest. Clinical trial registered with www.clinicaltrials.gov (NCT 00608764).

MicroRNA-149 inhibits PARP-2 and promotes mitochondrial biogenesis via SIRT-1/PGC-1α network in skeletal muscle.

High-fat diet (HFD) plays a central role in the initiation of mitochondrial dysfunction that significantly contributes to skeletal muscle metabolic disorders in obesity. However, the mechanism by which HFD weakens skeletal muscle metabolism by altering mitochondrial function and biogenesis is unknown. Given the emerging roles of microRNAs (miRNAs) in the regulation of skeletal muscle metabolism, we sought to determine whether activation of a specific miRNA pathway would rescue the HFD-induced mitochondrial dysfunction via the sirtuin-1 (SIRT-1)/ peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) pathway, a pathway that governs genes necessary for mitochondrial function. We here report that miR-149 strongly controls SIRT-1 expression and activity. Interestingly, miR-149 inhibits poly(ADP-ribose) polymerase-2 (PARP-2) and so increased cellular NAD+ levels and SIRT-1 activity that subsequently increases mitochondrial function and biogenesis via PGC-1α activation. In addition, skeletal muscles from HFD-fed obese mice exhibit low levels of miR-149 and high levels of PARP-2, and they show reduced mitochondrial function and biogenesis due to a decreased activation of the SIRT-1/PGC-1α pathway, suggesting that mitochondrial dysfunction in the skeletal muscle of obese mice may be because of, at least in part, miR-149 dysregulation. Overall, miR-149 may be therapeutically useful for treating HFD-induced skeletal muscle metabolic disorders in such pathophysiological conditions as obesity and type 2 diabetes.

Nebulette Mutations are Associated with Dilated Cardiomyopathy and Endocardial Fibroelastosis

OBJECTIVES Four variants (K60N, Q128R, G202R, and A592E) in the nebulette gene were identified in patients with dilated cardiomyopathy (DCM) and endocardial fibroelastosis. We sought to determine if these mutations are cardiomyopathy causing. BACKGROUND Nebulette aligns thin filaments and connects them with the myocardial Z-disk, playing a role in mechanosensation. METHODS We generated transgenic mice with cardiac-restricted overexpression of human wild-type or mutant nebulette. Chimera and transgenic mice were examined at 4, 6, and 12 months of age by echocardiography and cardiac magnetic resonance imaging. The hearts from embryos and adult mice were assessed by histopathologic, immunohistochemical, ultrastructural, and protein analyses. Rat H9C2 cardiomyoblasts with transient expression of nebulette underwent cyclic mechanical strain. RESULTS We identified lethal cardiac structural abnormalities in mutant embryonic hearts (K60N and Q128R). Founders of the mutant mouse lines developed DCM with severe heart failure. An irregular localization pattern for nebulette and impaired desmin expression were noted in the proband and chimeric Q128R mice. Mutant G202R and A592E mice exhibited left ventricular dilation and impaired function with specific changes in I-band and Z-disk proteins by 6 months of age. The mutations modulated distribution of nebulette in the sarcomere and Z-disk during stretch of H9C2 cells. CONCLUSIONS Nebulette is a new susceptibility gene for endocardial fibroelastosis and DCM. Different mutations in nebulette trigger specific mechanisms, converging to a common pathological cascade leading to endocardial fibroelastosis and DCM.

Induction of Sirt1 by Mechanical Stretch of Skeletal Muscle through the Early Response Factor EGR1 Triggers an Antioxidative Response

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species (1, 2). The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription (3–5). In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.