• Cell Membrane Disruption Stimulates NO/PKG Signaling and Potentiates Cell Membrane Repair in Neighboring Cells

      Togo, Tatsuru; McNeil, Paul L.; Department of Cellular Biology and Anatomy (2012-08-7)
      Resealing of a disrupted plasma membrane at the micron-diameter range requires Ca2+-regulated exocytosis. Repeated membrane disruptions reseal more quickly than the initial wound, and this potentiation of membrane resealing persists for at least 24 hours after the initial wound. Long-term potentiation of membrane resealing requires CREB-dependent gene expression, which is activated by the PKC- and p38 MAPK-dependent pathway in a wounded cell. The present study demonstrates that membrane resealing is potentiated in both wounded and neighboring cells in MDCK cells. Wounding of cells expressing CREB133, a mutant variant of CREB, does not show the potentiated response of cell membrane resealing in either wounded or neighboring cells. Furthermore, wounding of cells induces CREB phosphorylation, not only in wounded cells, but also in neighboring cells. Inhibition of the nitric oxide/PKG signaling pathway suppresses CREB phosphorylation in neighboring cells, but not in wounded cells. The potentiation of membrane resealing in neighboring cells is suppressed if the nitric oxide/PKG pathway is inhibited during the initial wound. Together, these results suggest that the nitric oxide/PKG pathway stimulates CREB phosphorylation in neighboring cells so that subsequent cell membrane disruptions of the neighboring cells reseal more quickly.
    • Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

      Ashley, Jason W.; Shi, Zhenqi; Zhao, Haibo; Li, Xingsheng; Kesterson, Robert A.; Feng, Xu; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-10-3)
      CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68â /â osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68â /â osteoclasts.
    • Human Platelet-Rich Plasma- and Extracellular Matrix-Derived Peptides Promote Impaired Cutaneous Wound Healing In Vivo

      Demidova-Rice, Tatiana N.; Wolf, Lindsey; Deckenback, Jeffry; Hamblin, Michael R.; Herman, Ira M.; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2012-02-23)
      Previous work in our laboratory has described several pro-angiogenic short peptides derived from endothelial extracellular matrices degraded by bacterial collagenase. Here we tested whether these peptides could stimulate wound healing in vivo. Our experiments demonstrated that a peptide created as combination of fragments of tenascin X and fibrillin 1 (comb1) applied into cranial dermal wounds created in mice treated with cyclophosphamide to impair wound healing, can improve the rate of wound closure. Furthermore, we identify and characterize a novel peptide (UN3) created and modified from two naturally-occurring peptides, which are present in human platelet-rich plasma. In vitro testing of UN3 demonstrates that it causes a 50% increase in endothelial proliferation, 250% increase in angiogenic response and a tripling of epithelial cell migration in response to injury.
    • Mitochondrial Dysfunction and Adipogenic Reduction by Prohibitin Silencing in 3T3-L1 Cells

      Liu, Dong; Lin, Yiming; Kang, Ting; Huang, Bo; Xu, Wei; Garcia-Barrio, Minerva; Olatinwo, Moshood; Matthews, Roland; Chen, Yuqing Eugene; Thompson, Winston E.; et al. (2012-03-30)
      Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.
    • Myostatin Is Elevated in Congenital Heart Disease and After Mechanical Unloading

      Bish, Lawrence T.; George, Isaac; Maybaum, Simon; Yang, Jonathan; Chen, Jonathan M.; Sweeney, H. Lee; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-09-13)
      Background: Myostatin is a negative regulator of skeletal muscle mass whose activity is upregulated in adult heart failure (HF); however, its role in congenital heart disease (CHD) is unknown.
    • Palmitoleate Induces Hepatic Steatosis but Suppresses Liver Inflammatory Response in Mice

      Guo, Xin; Li, Honggui; Xu, Hang; Halim, Vera; Zhang, Weiyu; Wang, Huan; Ong, Kuok Teong; Woo, Shih-Lung; Walzem, Rosemary L.; Mashek, Douglas G.; et al. (2012-06-29)
      The interaction between fat deposition and inflammation during obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD). The present study examined the effects of palmitoleate, a monounsaturated fatty acid (16⠶1n7), on liver metabolic and inflammatory responses, and investigated the mechanisms by which palmitoleate increases hepatocyte fatty acid synthase (FAS) expression. Male wild-type C57BL/6J mice were supplemented with palmitoleate and subjected to the assays to analyze hepatic steatosis and liver inflammatory response. Additionally, mouse primary hepatocytes were treated with palmitoleate and used to analyze fat deposition, the inflammatory response, and sterol regulatory element-binding protein 1c (SREBP1c) activation. Compared with controls, palmitoleate supplementation increased the circulating levels of palmitoleate and improved systemic insulin sensitivity. Locally, hepatic fat deposition and SREBP1c and FAS expression were significantly increased in palmitoleate-supplemented mice. These pro-lipogenic events were accompanied by improvement of liver insulin signaling. In addition, palmitoleate supplementation reduced the numbers of macrophages/Kupffer cells in livers of the treated mice. Consistently, supplementation of palmitoleate decreased the phosphorylation of nuclear factor kappa B (NF-κB, p65) and the expression of proinflammatory cytokines. These results were recapitulated in primary mouse hepatocytes. In terms of regulating FAS expression, treatment of palmitoleate increased the transcription activity of SREBP1c and enhanced the binding of SREBP1c to FAS promoter. Palmitoleate also decreased the phosphorylation of NF-κB p65 and the expression of proinflammatory cytokines in cultured macrophages. Together, these results suggest that palmitoleate acts through dissociating liver inflammatory response from hepatic steatosis to play a unique role in NAFLD.
    • Rhabdomyosarcomas in Aging A/J Mice

      Sher, Roger B.; Cox, Gregory A.; Mills, Kevin D.; Sundberg, John P.; McNeil, Paul L.; Department of Cellular Biology and Anatomy; College of Graduate Studies (2011-08-10)
      Rhabdomyosarcomas (RSCs) are skeletal muscle neoplasms found in humans and domestic mammals. The A/J inbred strain developed a high frequency (between 70â 80%) of adult pleomorphic type (APT) RSC at >20 months of age while BALB/cByJ also develop RSC but less frequently. These neoplasms invaded skeletal muscle surrounding either the axial or proximal appendicular skeleton and were characterized by pleomorphic cells with abundant eosinophilic cytoplasm, multiple nuclei, and cross striations. The diagnosis was confirmed by detection of alpha-sarcomeric actin and myogenin in the neoplastic cells using immunocytochemistry. The A/J strain, but not the related BALB/c substrains, is also characterised by a progressive muscular dystrophy homologous to limb-girdle muscular dystrophy type 2B. The association between the development of RSC in similar muscle groups to those most severely affected by the progressive muscular dystrophy suggested that these neoplasms developed from abnormal regeneration of the skeletal muscle exacerbated by the dysferlin mutation. Transcriptome analyses of RSCs revealed marked downregulation of genes in muscular development and function signaling networks. Non-synonymous coding SNPs were found in Myl1, Abra, Sgca, Ttn, and Kcnj12 suggesting these may be important in the pathogenesis of RSC. These studies suggest that A strains of mice can be useful models for dissecting the molecular genetic basis for development, progression, and ultimately for testing novel anticancer therapeutic agents dealing with rhabdomyosarcoma.