Faculty Bibliography

OBJECTIVE: To determine the role of ANK/Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes. METHOD: ANK/MYBBP1a and SPHK1 interactions were identified using yeast two-hybrid screening and co-immunoprecipitation. To determine the role of these interactions in catabolic events of articular chondrocytes, ank/ank and wild type mouse chondrocytes transfected with full-length or mutant ank expression vectors or femoral heads were treated with interleukin-1beta (IL-1beta) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin O staining and MMP-13 immunostaining were determined in femoral head explants; NF-kappaB activity was determined by transfecting chondrocytes with a NF-kappaB-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. RESULTS: The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-kappaB activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1beta-treated articular chondrocytes or femoral heads. Transfection with full-length ank expression vector reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-kappaB activities in IL-1beta-treated ank/ank chondrocytes, whereas transfection with P5L or F376del mutant ank reduced nuclear MYBBP1a or increased SPHK activity, respectively, and consequently either transfection only partially restored NF-kappaB activity. CONCLUSION: ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1beta-mediated cartilage degradation.

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2alpha), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor alpha (GM-CSFRalpha)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

Statins are the first line pharmacotherapy for cholesterol reduction. Use of these drugs in large, randomized clinical trials have consistently shown significant reductions in major vascular events including death, myocardial infarction, stroke, and coronary revascularization. The updated guidelines for the treatment of high blood cholesterol from the ACC/AHA, will lead to a rise in the number of patients taking statins. Hence, statin intolerance may subsequently increase, emphasizing the need to understand and treat this important problem.Clinical Pharmacology & Therapeutics (2014); Accepted article preview online 11 April 2014; doi:10.1038/clpt.2014.84.

Regulated messenger RNA (mRNA) decay is an essential mechanism that governs proper control of gene expression. In fact, many of the most physiologically potent proteins are encoded by short-lived mRNAs, many of which contain AU-rich elements (AREs) in their 3'-untranslated region (3'-UTR). AREs target mRNAs for post-transcriptional regulation, generally rapid decay, but also stabilization and translation inhibition. AREs control mRNA turnover and translation activities through association with trans-acting RNA-binding proteins that display high affinity for these AU-rich regulatory elements. AU-rich element RNA-binding protein (AUF1), also known as heterogeneous nuclear ribonucleoprotein D (HNRNPD), is an extensively studied AU-rich binding protein (AUBP). AUF1 has been shown to regulate ARE-mRNA turnover, primarily functioning to promote rapid ARE-mRNA degradation. In certain cellular contexts, AUF1 has also been shown to regulate gene expression at the translational and even the transcriptional level. AUF1 comprises a family of four related protein isoforms derived from a common pre-mRNA by differential exon splicing. AUF1 isoforms have been shown to display multiple and distinct functions that include the ability to target ARE-mRNA stability or decay, and transcriptional activation of certain genes that is controlled by their differential subcellular locations, expression levels, and post-translational modifications. AUF1 has been implicated in controlling a variety of physiological functions through its ability to regulate the expression of numerous mRNAs containing 3'-UTR AREs, thereby coordinating functionally related pathways. This review highlights the physiological functions of AUF1-mediated regulation of mRNA and gene expression, and the consequences of deficient AUF1 levels in different physiological settings. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.

The major risks of pacemaker and implantable cardioverter defibrillator extraction are attributable to the fibrotic tissue that encases them in situ, yet little is known about the cellular and functional properties of this response. In the present research, we performed a histological and mechanical analysis of human tissue collected from the lead-tissue interface to better understand this process and provide insights for the improvement of lead design and extraction. The lead-tissue interface consisted of a thin cellular layer underlying a smooth, acellular surface, followed by a circumferentially organized collagen-rich matrix. 51.8+/-4.9% of cells were myofibroblasts via immunohistochemistry, with these cells displaying a similar circumferential organization. Upon mechanical testing, samples exhibited a triphasic force-displacement response consisting of a toe region during initial tensioning, a linear elastic region and a yield and failure region. Mean fracture load was 5.6+/-2.1N, and mean circumferential stress at failure was 9.5+/-4.1MPa. While the low cellularity and fibrotic composition of tissue observed herein is consistent with a foreign body reaction to an implanted material, the significant myofibroblast response provides a mechanical explanation for the contractile forces complicating extractions. Moreover, the tensile properties of this tissue suggest the feasibility of circumferential mechanical tissue disruption, similar to balloon angioplasty devices, as a novel approach to assist with lead extraction.

Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.

Mitochondrial respiratory chain disorders are characterized by loss of electron transport chain (ETC) activity. Although the causes of many such diseases are known, there is a lack of effective therapies. To identify genes that confer resistance to severe ETC dysfunction when inactivated, we performed a genome-wide genetic screen in haploid human cells with the mitochondrial complex III inhibitor antimycin. This screen revealed that loss of ATPIF1 strongly protects against antimycin-induced ETC dysfunction and cell death by allowing for the maintenance of mitochondrial membrane potential. ATPIF1 loss protects against other forms of ETC dysfunction and is even essential for the viability of human rho degrees cells lacking mitochondrial DNA, a system commonly used for studying ETC dysfunction. Importantly, inhibition of ATPIF1 ameliorates complex III blockade in primary hepatocytes, a cell type afflicted in severe mitochondrial disease. Altogether, these results suggest that inhibition of ATPIF1 can ameliorate severe ETC dysfunction in mitochondrial pathology.

Kindlin-1 is an integrin tail binding protein that controls integrin activation. Mutations in the FERMT-1 gene, which encodes for Kindlin-1, lead to Kindler syndrome in man, which is characterized by skin blistering, premature skin aging and skin cancer of unknown etiology. Here we show that loss of Kindlin-1 in mouse keratinocytes recapitulates Kindler syndrome and also produces enlarged and hyperactive stem cell compartments, which lead to hyperthickened epidermis, ectopic hair follicle development and increased skin tumor susceptibility. Mechanistically, Kindlin-1 controls keratinocyte adhesion through beta1-class integrins and proliferation and differentiation of cutaneous epithelial stem cells by promoting alphavbeta6 integrin-mediated transforming growth factor-beta (TGF-beta) activation and inhibiting Wnt-beta-catenin signaling through integrin-independent regulation of Wnt ligand expression. Our findings assign Kindlin-1 the previously unknown and essential task of controlling cutaneous epithelial stem cell homeostasis by balancing TGF-beta-mediated growth-inhibitory signals and Wnt-beta-catenin-mediated growth-promoting signals.