Villin-1 and Gelsolin Regulate Changes in Actin Dynamics That Affect Cell Survival Signaling Pathways and Intestinal Inflammation.

BACKGROUND & AIMS: Cell stress signaling pathways result in phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (EIF2S1 or EIF2A), which affects regulation of protein translation. Translation reprogramming mitigates stress by activating pathways that result in autophagy and cell death, to eliminate damaged cells. Actin is modified during stress and EIF2A is dephosphorylated to restore homeostasis. It is not clear how actin affects EIF2A signaling. We studied the actin-binding proteins villin 1 (VIL1) and gelsolin (GSN) in intestinal epithelial cells (IECs) to determine whether they respond to cell stress response and affect signaling pathways. METHODS: We performed studies with mice with disruptions in Vil1 and Gsn (double-knockout mice). Wild-type (WT) mice either were or were not (controls) exposed to cell stressors such as tumor necrosis factor and adherent-invasive Escherichia coli. Distal ileum tissues were collected from mice; IECs and enteroids were cultured and analyzed by histology, immunoblots, phalloidin staining, immunohistochemistry, electron microscopy, and flow cytometry. HT-29 cells were incubated with cell stressors such as DTT, IFN, and adherent-invasive E coli or control agents; cells were analyzed by immunoblots and quantitative polymerase chain reaction. Green fluorescent protein and green fluorescent protein tagged mutant EIF2A were expressed from a lentiviral vector. The mouse immunity-related GTPase (IRGM1) was overexpressed in embryonic fibroblasts from dynamin1 like (DNM1L) protein-knockout mice or their WT littermates. IRGM1 was overexpressed in embryonic fibroblasts from receptor interacting serine/threonine kinase 1-knockout mice or their WT littermates. Human IRGM was overexpressed in human epithelial cell lines incubated with the DNM1L-specific inhibitor Mdivi-1. Mitochondria were analyzed by semi-quantitative confocal imaging. We performed immunohistochemical analyses of distal ileum tissues from 6-8 patients with Crohn's disease (CD) and 6-8 individuals without CD (controls). RESULTS: In IECs exposed to cell stressors, EIF2A signaling reduced expression of VIL1 and GSN. However, VIL1 and GSN were required for dephosphorylation of EIF2A and recovery from cell stress. In mouse and human IECs, prolonged, unresolved stress was accompanied by continued down-regulation of VIL1 and GSN, resulting in constitutive phosphorylation of EIF2A and overexpression of IRGM1 (or IRGM), which regulates autophagy. Overexpression of IRGM1 (or IRGM) induced cell death by necroptosis, accompanied by release of damage-associated molecular patterns (DAMPs). In double-knockout mice, constitutive phosphorylation of EIF2A and over-expression of IRGM1 resulted in spontaneous ileitis that resembled human CD in symptoms and histology. Distal ileum tissues from patients with CD had lower levels of VIL1 and GSN, increased phosphorylation of EIF2A, increased levels of IRGM and necroptosis, and increased release of nuclear DAMPs compared with controls. CONCLUSIONS: In studies of intestinal epithelial tissues from patients with CD and embryonic fibroblasts from mice, along with enteroids and human IEC lines, we found that induction of cell stress alters the cytoskeleton in IECs via changes in the actin-binding proteins VIL1 and GSN. Acute changes in actin dynamics increase IEC survival, whereas long-term changes in actin dynamics lead to IEC death and intestinal inflammation. IRGM regulates necroptosis and release of DAMPs to induce gastrointestinal inflammation, linking IRGM activity with CD.

Mutant p53 Promotes Epithelial Ovarian Cancer by Regulating Tumor Differentiation, Metastasis, and Responsiveness to Steroid Hormones.

Mutations in the tumor protein p53 (TP53) are the most frequently occurring genetic events in high-grade ovarian cancers, especially the prevalence of the Trp53(R172H)-mutant allele. In this study, we investigated the impact of the Trp53(R172H)-mutant allele on epithelial ovarian cancer (EOC) in vivo We used the Pten/Kras(G12D)-mutant mouse strain that develops serous EOC with 100% penetrance to introduce the mutant Trp53(R172H) allele (homolog for human Trp53(R172H)). We demonstrate that the Trp53(R172H) mutation promoted EOC but had differential effects on disease features and progression depending on the presence or absence of the wild-type (WT) TP53 allele. Heterozygous WT/Trp53(R172H) alleles facilitated invasion into the ovarian stroma, accelerated intraperitoneal metastasis, and reduced TP53 transactivation activity but retained responsiveness to nutlin-3a, an activator of WT TP53. Moreover, high levels of estrogen receptor α in these tumors enhanced the growth of both primary and metastatic tumors in response to estradiol. Ovarian tumors homozygous for Trp53(R172H) mutation were undifferentiated and highly metastatic, exhibited minimal TP53 transactivation activity, and expressed genes with potential regulatory functions in EOC development. Notably, heterozygous WT/Trp53(R172H) mice also presented mucinous cystadenocarcinomas at 12 weeks of age, recapitulating human mucinous ovarian tumors, which also exhibit heterozygous TP53 mutations (∼50%-60%) and KRAS mutations. Therefore, we present the first mouse model of mucinous tumor formation from ovarian cells and supporting evidence that mutant TP53 is a key regulator of EOC progression, differentiation, and responsiveness to steroid hormones. Cancer Res; 76(8); 2206-18. ©2016 AACR.

Comparison of Cesium-137 and X-ray Irradiators by Using Bone Marrow Transplant Reconstitution in C57BL/6J Mice.

Mice are used extensively in transplantation studies involving bone marrow ablation. Due to the increasing security issues and expenses involved with γ irradiators, self-contained X-ray irradiators have been increasing in popularity. We hypothesized that bone marrow ablation by irradiation of mice with a (137)Cs irradiator would be comparable to that from an X-ray source irradiator. A lethal-dose curve was obtained by irradiating C57BL/6J mice with 500, 700, 900, and 1100 cGy from either source. These data were used to determine the lethal radiation exposure range for a noncompetitive bone marrow engraftment curve for each source. At 90 d after reconstitution, the bone marrow engraftment curves revealed significant differences between the 2 sources in the establishment of B cell, myeloid, and T cell lineages. Murine B cell reconstitution after exposure to a (137)Cs source was greater than that after X-ray exposure at each dose level, whereas the converse was true for myeloid cell reconstitution. At the 1050- and 1100-cGy doses, mice irradiated by using the X-ray source demonstrated higher levels of T cell reconstitution but decreased survival compared with mice irradiated with the (137)Cs source. We concluded that although both sources ablated endogenous bone marrow sufficiently to enable stem cell engraftment, there are distinct physiologic responses that should be considered when choosing the optimal source for use in a study and that irradiation from the (137)Cs source was associated with lower overall morbidity due to opportunistic infection.

Effects of route of inoculation and viral genetic variation on antibody responses to polyomavirus SV40 in Syrian golden hamsters.

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation-but not viral genetic variation-is an important factor in production of neutralizing antibody to SV40.

Suprapubic bladder catheterization of male spinal-cord-injured Sprague-Dawley rats.

The rat spinal-cord-injury (SCI) model is widely used to study the pathologic mechanisms that contribute to sensory and motor dysfunction in humans. This model is thought to mimic many of the negative outcomes experienced by humans after spinal contusion injury. We theorized that manual bladder expression contributed to the kidney and bladder lesions reported in previous studies using the rat SCI model. In the present study, rats were surgically implanted with bladder catheters after spinal contusion injury to provide continuous drainage of urine. After 72 h, the rats were euthanized and their kidneys and bladders examined histologically. BUN, serum creatinine, and urine protein were compared at 0 and 72 h after surgery. Kidney and bladder lesions were similar in SCI rats with and without implanted bladder catheters. BUN at 72 h was higher than baseline values in both groups, whereas serum creatinine was higher at 72 h compared with baseline values only in the catheterized rats. These findings indicate that suprapubic bladder catheterization does not reduce hydronephrosis in SCI rats and that the standard of care for bladder evacuation should continue to be manual expression of urine.

Disrupting circadian homeostasis of sympathetic signaling promotes tumor development in mice.

BACKGROUND: Cell proliferation in all rapidly renewing mammalian tissues follows a circadian rhythm that is often disrupted in advanced-stage tumors. Epidemiologic studies have revealed a clear link between disruption of circadian rhythms and cancer development in humans. Mice lacking the circadian genes Period1 and 2 (Per) or Cryptochrome1 and 2 (Cry) are deficient in cell cycle regulation and Per2 mutant mice are cancer-prone. However, it remains unclear how circadian rhythm in cell proliferation is generated in vivo and why disruption of circadian rhythm may lead to tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: Mice lacking Per1 and 2, Cry1 and 2, or one copy of Bmal1, all show increased spontaneous and radiation-induced tumor development. The neoplastic growth of Per-mutant somatic cells is not controlled cell-autonomously but is dependent upon extracellular mitogenic signals. Among the circadian output pathways, the rhythmic sympathetic signaling plays a key role in the central-peripheral timing mechanism that simultaneously activates the cell cycle clock via AP1-controlled Myc induction and p53 via peripheral clock-controlled ATM activation. Jet-lag promptly desynchronizes the central clock-SNS-peripheral clock axis, abolishes the peripheral clock-dependent ATM activation, and activates myc oncogenic potential, leading to tumor development in the same organ systems in wild-type and circadian gene-mutant mice. CONCLUSIONS/SIGNIFICANCE: Tumor suppression in vivo is a clock-controlled physiological function. The central circadian clock paces extracellular mitogenic signals that drive peripheral clock-controlled expression of key cell cycle and tumor suppressor genes to generate a circadian rhythm in cell proliferation. Frequent disruption of circadian rhythm is an important tumor promoting factor.

Scaffold Attachment Factor B1 (SAFB1) heterozygosity does not influence Wnt-1 or DMBA-induced tumorigenesis.

BACKGROUND: Scaffold Attachment Factor B1 (SAFB1) is a multifunctional protein which has been implicated in breast cancer previously. We recently generated SAFB1 knockout mice (SAFB1-/-), but pleiotropic phenotypes including high lethality, dwarfism associated with low IGF-I levels, and infertility and subfertility in male and female mice, respectively, do not allow for straightforward tumorigenesis studies in these mice. Therefore, we asked whether SAFB1 heterozygosity would influence tumor development and progression in MMTV-Wnt-1 oncomice or DMBA induced tumorigenicity, in a manner consistent with haploinsufficiency of the remaining allele. METHODS: We crossed female SAFB1+/- (C57B6/129) mice with male MMTV-Wnt-1 (C57B6/SJL) mice to obtain SAFB1+/+/Wnt-1, SAFB1+/-/Wnt-1, and SAFB1+/- mice. For the chemical induced tumorigenesis study we treated 8 weeks old SAFB1+/- and SAFB+/+ BALB/c mice with 1 mg DMBA once per week for 6 weeks. Animals were monitored for tumor incidence and tumor growth. Tumors were characterized by performing H&E, and by staining for markers of proliferation and apoptosis. RESULTS: We did not detect significant differences in tumor incidence and growth between SAFB1+/+/Wnt-1 and SAFB1+/-/Wnt-1 mice, and between DMBA-treated SAFB1+/+ and SAFB1+/-mice. Histological evaluation of tumors showed that SAFB1 heterozygosity did not lead to changes in proliferation or apoptosis. There were, however, significant differences in the distribution of tumor histologies with an increase in papillary and cribriform tumors, and a decrease in squamous tumors in the SAFB1+/-/Wnt-1 compared to the SAFB1+/+/Wnt-1 tumors. Of note, DMBA treatment resulted in shortened survival of SAFB1+/- mice compared to their wildtype littermates, however this trend did not reach statistical significance. CONCLUSION: Our data show that SAFB1 heterozygosity does not influence Wnt-1 or DMBA-induced mammary tumorigenesis.

MMP13, Birc2 (cIAP1), and Birc3 (cIAP2), amplified on chromosome 9, collaborate with p53 deficiency in mouse osteosarcoma progression.

Osteosarcoma is the primary malignant cancer of bone and particularly affects adolescents and young adults, causing debilitation and sometimes death. As a model for human osteosarcoma, we have been studying p53(+/-) mice, which develop osteosarcoma at high frequency. To discover genes that cooperate with p53 deficiency in osteosarcoma formation, we have integrated array comparative genomic hybridization, microarray expression analyses in mouse and human osteosarcomas, and functional assays. In this study, we found seven frequent regions of copy number gain and loss in the mouse p53(+/-) osteosarcomas but have focused on a recurrent amplification event on mouse chromosome 9A1. This amplicon is syntenic with a similar chromosome 11q22 amplicon identified in several human tumor types. Three genes on this amplicon, the matrix metalloproteinase gene MMP13 and the antiapoptotic genes Birc2 (cIAP1) and Birc3 (cIAP2), show elevated expression in mouse and human osteosarcomas. We developed a functional assay using clonal osteosarcoma cell lines transduced with lentiviral short hairpin RNA vectors to show that down-regulation of MMP13, Birc2, or Birc3 resulted in reduced tumor growth when transplanted into immunodeficient recipient mice. These experiments revealed that high MMP13 expression enhances osteosarcoma cell survival and that Birc2 and Birc3 also enhance cell survival but only in osteosarcoma cells with the chromosome 9A1 amplicon. We conclude that the antiapoptotic genes Birc2 and Birc3 are potential oncogenic drivers in the chromosome 9A1 amplicon.

SV40 lymphomagenesis in Syrian golden hamsters.

Simian virus 40 (SV40) isolates differ in oncogenic potential in Syrian golden hamsters following intraperitoneal inoculation. Here we describe the effect of intravenous exposure on tumor induction by SV40. Strains SVCPC (simple regulatory region) and VA45-54(2E) (complex regulatory region) were highly oncogenic following intravenous inoculation, producing a spectrum of tumor types. Three lymphoma cell lines were established; all expressed SV40 T-antigen, were immortalized for growth in culture, and were tumorigenic following transplantation in vivo. New monoclonal antibodies directed against hamster lymphocyte surface antigens are described. The cell lines expressed MHC class II and macrophage markers and were highly phagocytic, indicating a histiocytic origin. Many hamsters that remained tumor-free developed SV40 T-antigen antibodies, suggesting that viral replication occurred. This study shows that route of exposure influences the pathogenesis of SV40-mediated carcinogenesis, that SV40 strain VA45-54(2E) is lymphomagenic in hamsters, that hamster lymphoid cells of histiocytic origin can be transformed in vivo and established in culture, and that reagents to hamster leukocyte differentiation molecules are now available.

Activation of MAPK pathways links LMNA mutations to cardiomyopathy in Emery-Dreifuss muscular dystrophy.

Mutations in LMNA, which encodes nuclear Lamins A and C cause diseases affecting various organs, including the heart. We have determined the effects of an Lmna H222P mutation on signaling pathways involved in the development of cardiomyopathy in a knockin mouse model of autosomal dominant Emery-Dreifuss muscular dystrophy. Analysis of genome-wide expression profiles in hearts using Affymetrix GeneChips showed statistically significant differences in expression of genes in the MAPK pathways at the incipience of the development of clinical disease. Using real-time PCR, we showed that activation of MAPK pathways preceded clinical signs or detectable molecular markers of cardiomyopathy. In heart tissue and isolated cardiomyocytes, there was activation of MAPK cascades and downstream targets, implicated previously in the pathogenesis of cardiomyopathy. Expression of H222P Lamin A in cultured cells activated MAPKs and downstream target genes. Activation of MAPK signaling by mutant A-type lamins could be a cornerstone in the development of heart disease in autosomal dominant Emery-Dreifuss muscular dystrophy.

Pathology and nuclear abnormalities in hearts of transgenic mice expressing M371K lamin A encoded by an LMNA mutation causing Emery-Dreifuss muscular dystrophy.

Mutations in LMNA, which encodes nuclear lamins A and C, cause a broad range of diseases, including autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) and related disorders with a predominant cardiomyopathy. Homozygous Lmna model "knock-in" and null mice develop cardiomyopathy, whereas heterozygous mice do not. Overexpression of lamin A mutants that cause cardiomyopathy in cultured cells induces morphological abnormalities in the nuclear envelope and lamina; however, effects on tissue and organ pathology have not been determined. We used the heart-selective alpha-myosin heavy chain promoter to drive expression in transgenic mice of human wild-type and M371K lamin A, which causes EDMD. Mice expressing M371K lamin A were born at approximately 0.07 of the expected frequency and those born typically died at 2-7 weeks of age. Histological analysis showed increased eosinophilia and fragmentation of cardiomyofibrils, nuclear pyknosis and edema without fibrosis or significant inflammation, indicative of acute or subacute injury. Mice expressing human wild-type lamin A were born at only slightly less than the expected frequency and had normal life spans. Confocal immunofluorescence microscopy demonstrated abnormal nuclear envelopes with intranuclear foci of lamins in cardiac cells expressing M371K lamin A. Electron microscopy revealed extensively convoluted nuclear envelopes, intranuclear inclusions and chromatin clumps in cardiomyocyte nuclei. These results demonstrate that expression of a lamin A mutant that induces alterations in nuclear morphology can cause tissue and organ damage in mice with a normal complement of wild-type lamins.

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers.

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Social environment influences the progression of atherosclerosis in the watanabe heritable hyperlipidemic rabbit.

BACKGROUND: Although there is evidence that emotionally stressful behavior can accelerate the progression of atherosclerosis, there is less data to support the notion that affiliative social behavior can slow disease progression. The present study examines the influence of social environment on the progression of atherosclerosis in the Watanabe Heritable Hyperlipidemic (WHHL) rabbit, a model that spontaneously develops lesions because of a genetic defect in lipoprotein clearance. METHODS AND RESULTS: WHHL rabbits were assigned to 1 of 3 social or behavioral groups: an unstable group, in which unfamiliar rabbits were paired daily, with the pairing switched each week; a stable group, in which littermates were paired daily for the entire study; and an individually caged group. The stable group exhibited more affiliative social behavior and less agonistic behavior than the unstable group and significantly less aortic atherosclerosis than each of the other 2 groups. Although the unstable and individually caged groups had comparable aortic lesion areas, the severity of the disease progressed faster in the unstable group, as indexed by a larger area of calcification and increased fibrous cap thickness in complex lesions. The unstable group showed increased agonistic behavior and signs of chronic adrenocortical and gonadal activation, whereas the individually caged group was relatively sedentary, had low glucocorticoid levels, and was hyperinsulinemic compared with the other groups. CONCLUSIONS: The present study demonstrates that social environment can slow, as well as accelerate, the progression of atherosclerosis. It also emphasizes the importance of behavioral factors in atherogenesis, even in a model of disease with strong genetic determinants.