Deletion of Ulk1 inhibits neointima formation by enhancing KAT2A/GCN5-mediated acetylation of TUBA/α-tubulin in vivo

AUTHORS

Changhan Ouyang, Jian Li, Xiaoxu Zheng, Jing Mu, Gloria Torres, Qilong Wang, Ming-Hui Zou & Zhonglin Xie

ABSTRACT

ULK1 (unc-51 like autophagy activating kinase) has a central role in initiating macroautophagy/autophagy, a process that contributes to atherosclerosis and neointima hyperplasia, or excessive tissue growth that leads to vessel dysfunction. However, the role of ULK1 in neointima formation remains unclear. We aimed to determine how Ulk1 deletion affected neointima formation and to investigate the underlying mechanisms. We measured autophagy activity, vascular smooth muscle cell (VSMC) migration and neointima hyperplasia in cultured VSMCs and ligation-injured mouse carotid arteries from male wild-type (WT, C57BL/6 J) and VSMC-specific ulk1 knockout (ulk1 KO) mice. Carotid artery ligation in WT mice increased ULK1 protein expression, and concurrently increased autophagic flux and neointima formation. Treating human aortic smooth muscle cells (HASMCs) with PDGF (platelet derived growth factor) increased ULK1 expression, activated autophagy, and promoted cell migration. Further, smooth muscle cell-specific deletion of Ulk1 suppressed autophagy, inhibited VSMC migration, and impeded neointima hyperplasia. Mechanistically, Ulk1 deletion inhibited autophagic degradation of histone acetyltransferase protein KAT2A/GCN5 (K[lysine] acetyltransferase 2A), resulting in accumulation of KAT2A that directly acetylated TUBA/α-tubulin and subsequently increased protein levels of acetylated TUBA. The acetylation of TUBA increased microtubule stability and inhibited VSMC directional migration and neointima formation. Finally, local transfection of Kat2a siRNA decreased TUBA acetylation and prevented the attenuation of vascular injury-induced neointima formation in ulk1 KO mice. These findings suggest that Ulk1 deletion inhibits neointima formation by reducing autophagic degradation of KAT2A and increasing TUBA acetylation in VSMCs.

Sestrin2 Regulates Osteoclastogenesis via the p62-TRAF6 Interaction

AUTHORS

Sue Young Oh, Namju Kang, Jung Yun Kang, Ki Woo Kim, Jong-Hoon Choi, Yu-Mi Yang1 and Dong Min Shin

ABSTRACT

The receptor activator of nuclear factor-kappa B ligand (RANKL) mediates osteoclast differentiation and functions by inducing Ca2+ oscillations, activating mitogen-activated protein kinases (MAPKs), and activating nuclear factor of activated T-cells type c1 (NFATc1) via the RANK and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interaction. Reactive oxygen species (ROS) also plays an important role during osteoclastogenesis and Sestrin2, an antioxidant, maintains cellular homeostasis upon stress injury via regulation of ROS, autophagy, and inflammation. However, the role of Sestrin2 in osteoclastogenesis remains unknown. In this study, we investigated the role of Sestrin2 in the RANKL-RANK-TRAF6 signaling pathway during osteoclast differentiation. Deletion of Sestrin2 (Sesn2) increased bone mass and reduced the number of multinucleated osteoclasts on bone surfaces. RANKL-induced osteoclast differentiation and function decreased in Sesn2 knockout (KO) bone marrow-derived monocytes/macrophages (BMMs) due to inhibition of NFATc1 expression, but osteoblastogenesis was not affected. mRNA expression of RANKL-induced specific osteoclastogenic genes and MAPK protein expression were lower in Sesn2 KO BMMs than wild-type (WT) BMMs after RANKL treatment. However, the Sesn2 deletion did not affect ROS generation or intracellular Ca2+ oscillations during osteoclastogenesis. In contrast, the interaction between TRAF6 and p62 was reduced during osteoclasts differentiation in Sesn2 KO BMMs. The reduction in the TRAF6/p62 interaction and TRAP activity in osteoclastogenesis in Sesn2 KO BMMs was recovered to the WT level upon expression of Flag-Sesn2 in Sesn2 KO BMMs. These results suggest that Sestrin2 has a novel role in bone homeostasis and osteoclasts differentiation through regulation of NFATc1 and the TRAF6/p62 interaction.

The quality of etched enamel in different regions and tooth types and its significance in bonding and the development of white spot lesions

AUTHORS

Elisabeth C. Barnhart; Phillip M. Campbell; Amal Noureldin; Katie Julien; Peter H. Buschang

ABSTRACT

Objectives

To quantify differences in the etch quality of enamel within and between human teeth, which has not previously been attempted.

Materials and Methods

The buccal right and left halves of 27 extracted human teeth were randomly allocated to scanning electron microscopy (SEM) or micro–computed tomography (μCT) for evaluation. The buccal surfaces were pumiced, etched with 37% phosphoric acid gel etchant for 15 seconds, rinsed, and air dried. Each tooth was divided into three regions (incisal, middle, and cervical) and viewed after etching at 1200× magnification with SEM. The μCT scans were taken before and after etching to calculate apparent and material mineral densities.

Results

SEM showed greater aprismatic enamel and poorer etch quality (ie, significantly less percentage enamel) for the posterior than anterior teeth and for the cervical region than for the incisal and middle regions of all teeth. Although there were no density differences prior to etching, μCT demonstrated that etching increased material density significantly more for the anterior than posterior teeth. Prior to etching, the enamel in the cervical regions was significantly less dense than the enamel in the middle or incisal regions. Etching significantly increased the material density of all three regions, which decreased initial regional differences. After etching, the apparent density of the cervical region remained significantly lower than the densities of the other two regions.

Conclusions

Based on SEM and μCT, there is greater aprismatic enamel and inferior etch quality in the cervical regions of all tooth types and is clinically significant in explaining the failure of sealant retention and the propensity for white spot lesions.

Homoarginine ameliorates diabetic nephropathy independent of nitric oxide synthase-3

AUTHORS

Michael D. Wetzel, Kristen Stanley, Soumya Maity, Muniswamy Madesh, Jean C. Bopassa, Alaa S. Awad

ABSTRACT

Recently we showed that homoarginine supplementation confers kidney protection in diabetic mouse models. In this study we tested whether the protective effect of homoarginine is nitric oxide synthase-3 (NOS3)-independent in diabetic nephropathy (DN). Experiments were conducted in NOS3 deficient (NOS3−/−) mice and their wild type littermate using multiple low doses of vehicle or streptozotocin and treated with homoarginine via drinking water for 24 weeks. Homoarginine supplementation for 24 weeks in diabetic NOS3−/− mice significantly attenuated albuminuria, increased blood urea nitrogen, histopathological changes and kidney fibrosis, kidney fibrotic markers, and kidney macrophage recruitment compared with vehicle-treated diabetic NOS3−/− mice. Furthermore, homoarginine supplementation restored kidney mitochondrial function following diabetes. Importantly, there were no significant changes in kidney NOS1 or NOS2 mRNA expression between all groups. In addition, homoarginine supplementation improved cardiac function and reduced cardiac fibrosis following diabetes. These data demonstrate that the protective effect of homoarginine is independent of NOS3, which will ultimately change our understanding of the mechanism(s) by which homoarginine induce renal and cardiac protection in DN. Homoarginine protective effect in DN could be mediated via improving mitochondrial function.

Protective effects of sirtuin 3 on titanium particle-induced osteogenic inhibition by regulating the NLRP3 inflammasome via the GSK-3β/β-catenin signalling pathway

AUTHORS

Kai Zheng, Jiaxiang Bai, Ning Li, Meng Li, Houyi Sun, Weicheng Zhang, Gaoran Ge, Xiaolong Liang, Huaqiang Tao, Yi Xue, Yuefeng Hao, Chen Zhu, Yaozeng Xu, Dechun Geng

ABSTRACT

Periprosthetic osteolysis (PPO) remains the key factor in implant failure and subsequent revision surgery and is mainly triggered by wear particles. Previous studies have shown that inhibition of osteoblastic differentiation is the most widespread incident affecting the interface of trabecular and loosening prostheses. Additionally, the NLRP3 inflammasome is activated by prosthetic particles. Sirtuin3, an NAD+-dependent deacetylase of mitochondria, regulates the function of mitochondria in diverse activities. However, whether SIRT3 can mitigate wear debris-induced osteolysis by inhibiting the NLRP3 inflammasome and enhancing osteogenesis has not been previously reported. Therefore, we investigated the role of SIRT3 during the process of titanium (Ti) particle-induced osteolysis. We revealed that upregulated SIRT3 dramatically attenuated Ti particle-induced osteogenic inhibition through suppression of the NLRP3 inflammasome and improvement of osteogenesis in vivo and in vitro. Moreover, we found that SIRT3 interference in the process of Ti particle-induced osteolysis relied on the GSK-3β/β-catenin signalling pathway. Collectively, these findings indicated that SIRT3 may serve as a rational new treatment against debris-induced PPO by deacetylase-dependent inflammasome attenuation.

Citrullinated vimentin mediates development and progression of lung fibrosis

AUTHORS

FU JUN LI, RANU SUROLIA, HUASHI LIZHENG WANG, GANG LIU, TEJASWINI KULKARNI, ADRIANA V. F. MASSICANO, JAMES A. MOBLEY, SANTANU MONDAL, JOAO A. DE ANDRADE, SCOTT A. COONROD, PAUL R. THOMPSON, KEITH WILLE, SUZANNE E. LAPI, MOHAMMAD ATHAR, VICTOR J. THANNICKAL, A. BRENT CARTER AND VEENA B. ANTONY

ABSTRACT

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)–dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-β1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2−/− and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.