Faculty Bibliography

Objective. To determine the actions of lithium chloride (LiCl) on catabolic events in articular chondrocytes and cartilage degradation after interleukin (IL)-1beta(beta) treatment and after surgically induced osteoarthritis (OA) in mice. Methods. The expression levels of catabolic genes in human articular chondrocytes treated with LiCl followed by IL-1beta were determined by real time PCR. To understand the mechanism of how LiCl affects catabolic events in articular chondrocytes after IL-1beta treatment, the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) was determined using luciferase reporter assays, and the activities of mitogen-activated protein kinases (MAPK) and signal transducer and activator of transcription 3 (Stat3) signaling pathway were determined by immunoblot analysis of total cell lysates. Mouse femoral head explant cultures treated with IL-1beta and a surgically induced OA mouse model were used to determine the effect of LiCl on proteoglycan loss and cartilage degradation. Results. LiCl treatment resulted in decreased catabolic marker mRNA levels and activation of NF-kappaB, p38 MAPK, and Stat3 signaling in IL-1beta -treated chondrocytes. Furthermore, LiCl directly inhibited IL-6-stimulated activation of Stat3 signaling. Consequently, loss of proteoglycan and cartilage destruction in LiCl-treated knee joints 8 weeks after OA-induced surgery or in LiCl-treated femoral head explants after IL-1beta treatment were markedly reduced compared to vehicle-treated joints or explants. Conclusion. LiCl reduced catabolic events in IL-1beta -treated human articular chondrocytes and cartilage destruction in IL-1beta -treated mouse femoral head explants and in surgically induced OA mouse models via the inhibition of NF-kappaB, p38 and Stat3 signaling pathway activities. (c) 2014 American College of Rheumatology.

Pancreatic ductal adenocarcinoma (PDAC) is strikingly resistant to conventional therapeutic approaches. We previously demonstrated that the histone deacetylase-associated protein SIN3B is essential for oncogene-induced senescence in cultured cells. Here, using a mouse model of pancreatic cancer, we have demonstrated that SIN3B is required for activated KRAS-induced senescence in vivo. Surprisingly, impaired senescence as the result of genetic inactivation of Sin3B was associated with delayed PDAC progression and correlated with an impaired inflammatory response. In murine and human pancreatic cells and tissues, levels of SIN3B correlated with KRAS-induced production of IL-1alpha. Furthermore, evaluation of human pancreatic tissue and cancer cells revealed that Sin3B was decreased in control and PDAC samples, compared with samples from patients with pancreatic inflammation. These results indicate that senescence-associated inflammation positively correlates with PDAC progression and suggest that SIN3B has potential as a therapeutic target for inhibiting inflammation-driven tumorigenesis.

Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing. Stem Cells 2014;32:1347-1360.

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.

Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

Introduction: The main function of connexins is to form gap junctions; yet, recent studies show that Cx43 is not only a gap junction protein. In fact, Cx43 is a part of a protein interacting network (the connexome), likely to regulate other functions in a gap junction-independent manner. Recently, it was reported that loss of the last five amino acids of Cx43 (Cx43D378stop) leads to lethal ventricular arrhythmias in mice. Localization of Cx43 at the membrane and electrical coupling between cells was normal. Interestingly, there was a significant loss of sodium current amplitude. These observations linked two fundamental steps in action potential propagation, excitability and electrical coupling, through a common molecular mechanism. Here, we explore the hypothesis that the microtubular network at the cell end is part of the common link. Methods: N/A Results: Functional assays: Macropatch, and super-resolution scanning patch clamp in ventricular myocytes isolated from Cx43D378stop and Cre-negative (control) mice revealed a reduction in the amplitude of sodium current exclusively at the intercalated disc (ID), without a change in channel unitary conductance. Super-resolution fluorescence microscopy: direct stochastic optical reconstruction microscopy (20 nm resolution) showed Nav1.5 clusters in close proximity (or overlapping) with N-cadherin plaques. The distance between NaV1.5 clusters and the cell end increased from 57.2+12nm, n=365 in control to 111.7+11nm, n=446 in Cx43D378stop myocytes (p<0.001), indicating that mutation Cx43D378stop reduced NaV1.5 surface expression. This coincided with separation of the microtubule plus-end protein EB1 from N-cadherin-rich cell ends, from 23.7+31.9nm, n=665 in control, to 123.5+13.5nm, n=502 in Cx43D378stop cells (p<0.05). Conclusions: Functional surface expression of NaV1.5 at the ID depends on preservation of the Cx43 C-end. Cx43 is part of a molecular complex that anchors the microtubule plus-end to the cell end, thus allowing proper delivery of its ca!