MERIT40 cooperates with BRCA2 to resolve DNA interstrand cross-links

MERIT40 is an essential component of the RAP80 ubiquitin recognition complex that targets BRCA1 to DNA damage sites. Although this complex is required for BRCA1 foci formation, its physiologic role in DNA repair has remained enigmatic, as has its relationship to canonical DNA repair mechanisms. Surprisingly, we found that Merit40^−/− mice displayed marked hypersensitivity to DNA interstrand cross-links (ICLs) but not whole-body irradiation. MERIT40 was rapidly recruited to ICL lesions prior to FANCD2, and Merit40-null cells exhibited delayed ICL unhooking coupled with reduced end resection and homologous recombination at ICL damage. Interestingly, Merit40 mutation exacerbated ICL-induced chromosome instability in the context of concomitant Brca2 deficiency but not in conjunction with Fancd2 mutation. These findings implicate MERIT40 in the earliest stages of ICL repair and define specific functional interactions between RAP80 complex-dependent ubiquitin recognition and the Fanconi anemia (FA)–BRCA ICL repair network.     

Isoform-specific p73 knockout mice reveal a novel role for ΔNp73 in the DNA damage response pathway

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and ΔNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the ΔNp73 isoform. Mice lacking ΔNp73 (ΔNp73^−/− mice) are viable and fertile but display signs of neurodegeneration. Cells from ΔNp73^−/− mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in ΔNp73^−/− cells, we discovered a completely new role for ΔNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that ΔNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of ΔNp73 expression show enhanced resistance to chemotherapy.     

Autocrine prolactin induced by the Pten–Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten–PI3K–Akt pathway. Conditional activation of the PI3K–Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K–Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)–Jak–Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl^−/−, Prlr^−/−, and Stat5^−/− mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1^−/−;Akt2^+/− mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K–Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K–Akt pathway.     

Pathways Contributing to Development of Spontaneous Mammary Tumors in BALB/c-Trp53+/− Mice

Mutation and loss of function in p53 are common features among human breast cancers. Here we use BALB/c-Trp53^+/− mice as a model to examine the sequence of events leading to mammary tumors. Mammary gland proliferation rates were similar in both BALB/c-Trp53^+/− mice and wild-type controls. In addition, sporadic mammary hyperplasias were rare in BALB/c-Trp53^+/− mice and not detectably different from those of wild-type controls. Among the 28 mammary tumors collected from BALB/c-Trp53^+/− mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53^+/− ductal hyperplasias also indicated an association between loss of the wild-type allele of Trp53 and progression to invasive carcinomas. Therefore, loss of p53 function seems to be a rate-limiting step in progression. Moreover, expression of biomarkers such as estrogen receptor α, progesterone receptor, Her2/Neu, and activated Notch1 varied among mammary tumors, suggesting that multiple oncogenic lesions collaborate with loss of p53 function. Expression of biomarkers was retained when tumor fragments were transplanted to syngeneic hosts. Tumors expressing solely luminal or basal keratins were also observed (27 and 11%, respectively), but the largest class of tumors expressed both luminal and basal keratins (62%). Overall, this panel of transplantable tumors provides a resource for detailed evaluation of the cell lineages undergoing transformation and preclinical testing of therapeutic agents targeting a variety of oncogenic pathways including cancer stem cells.Compromised function of the p53 tumor suppressor pathway remains among the most common alterations found in cancers.^1,2 Although disruption of p53 function predisposes to a broad spectrum of malignancies, the breast epithelium seems exquisitely sensitive to proper p53 function. Polymorphisms in MDM2, CHK2, and ATM alter the stability and activity of p53 and have been linked to breast cancer risk in women. The activity of p53 has also been shown to be responsive to hormones in rodent models.^3–5 Exogenous estrogen and progesterone are sufficient to render the mammary epithelium resistant to carcinogen-induced tumors, mimicking the protective effect afforded by a full-term pregnancy,⁶ and the p53 pathway participates in the hormone-induced protection.^7,8 Furthermore, breast cancer is the most prevalent tumor among women with Li-Fraumeni syndrome, which is most commonly associated with heterozygous mutations in TP53.^9–11 Reduced dosage of the p53 gene has been associated with haploinsufficiency with respect to levels of p53 protein and activity, cell cycle arrest, apoptosis, and homology-directed DNA repair.^12–14 Therefore, the level of p53 activity is a critical regulator of tumor suppressor pathways and breast cancer risk.The mechanisms by which p53 suppresses tumors are diverse. The roles of p53 in mediating cell cycle arrest and apoptosis have been described extensively.¹⁵ More recently, the activities of p53 have been expanded to include regulation of DNA repair, senescence, autophagy, cellular metabolism, and microRNA processing.^16–21 In addition, loss of p53 was shown to permit expansion of the pool of pluripotent embryonic stem cells^22,23 and cancer stem cells.^23–25 The activities of p53 that are critical for suppression of tumors vary among tissues. In the thymus, the proapoptotic activity of p53 was necessary to suppress lymphomas, whereas the cell cycle checkpoint function was dispensable.²⁶ In contrast, senescence is the principal pathway leading to regression of liver tumors after restoration of p53 function²⁷ and seems to be the prominent pathway in sarcomas as well.²⁸ Among the known breast cancer susceptibility genes there is a convergence of function highlighting the central role of homology-directed repair of DNA double-strand breaks in breast cancer risk.²⁹ Thus, fidelity of double-strand break repair may be a critical pathway controlled by p53 in breast tissue.The sequential changes that occur in normal tissue and in premalignant mammary lesions as a consequence of heterozygous mutations in TP53 provide clues to the mechanisms that initiate the carcinogenic cascade as well as the cellular origins of breast cancer. Although it is difficult to monitor sequential changes in human breast cancers, spontaneous mammary tumors are common in BALB/c-Trp53^+/− female mice,^30,31 providing a model to examine the sequence of phenotypic and genetic alterations during mammary tumorigenesis. Using this model, we demonstrate that heterozygosity for Trp53 does not increase proliferation of the epithelium or the incidence of precancerous lesions. However, loss of the wild-type allele of Trp53 was associated with the transition from hyperplastic to invasive phenotypes. In a set of 28 spontaneous tumors, histological phenotypes and expression of oncogenes were heterogeneous. The majority of tumors expressed markers of both luminal and basal epithelia (62%), suggesting that progenitor cells are the most common origin. However, significant numbers of tumors expressed purely luminal keratins (27%) or basal keratins (11%). Although distinct patterns of keratins were observed, stem cell markers were expressed similarly in tumors with only keratins associated with luminal cells (K8/18) as well as tumors expressing both luminal and basal cell keratins (K8/18 and K5/6). Therefore, it seems that tumors arise most frequently from progenitor cells, which then commit toward more differentiated lineages during progression. Lineage decisions were not associated with specific activation of either Notch1 or Her2. Because tumor phenotypes were stable after transplantation, this panel of tumors can be used to speed preclinical testing of chemotherapeutic agents.     

Satb1 and Satb2 regulate embryonic stem cell differentiation and Nanog expression

Satb1 and the closely related Satb2 proteins regulate gene expression and higher-order chromatin structure of multigene clusters in vivo. In examining the role of Satb proteins in murine embryonic stem (ES) cells, we find that Satb1^−/− cells display an impaired differentiation potential and augmented expression of the pluripotency determinants Nanog, Klf4, and Tbx3. Metastable states of self-renewal and differentiation competence have been attributed to heterogeneity of ES cells in the expression of Nanog. Satb1^−/− cultures have a higher proportion of Nanog^high cells, and an increased potential to reprogram human B lymphocytes in cell fusion experiments. Moreover, Satb1-deficient ES cells show an increased expression of Satb2, and we find that forced Satb2 expression in wild-type ES cells antagonizes differentiation-associated silencing of Nanog and enhances the induction of NANOG in cell fusions with human B lymphocytes. An antagonistic function of Satb1 and Satb2 is also supported by the almost normal differentiation potential of Satb1^−/−Satb2^−/− ES cells. Taken together with the finding that both Satb1 and Satb2 bind the Nanog locus in vivo, our data suggest that the balance of Satb1 and Satb2 contributes to the plasticity of Nanog expression and ES cell pluripotency.     

The scaffold protein WRAP53β orchestrates the ubiquitin response critical for DNA double-strand break repair

The WD40 domain-containing protein WRAP53β (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53β as an essential regulator of DNA double-strand break (DSB) repair. WRAP53β rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53β targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53β facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53β controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53β impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53β as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.     

β2 Integrin deficiency yields unconventional double-negative T cells distinct from mature classical natural killer T cells in mice

Expressed on leucocytes, β₂ integrins (CD11/CD18) are specifically involved in leucocyte function. Using a CD18-deficient (CD18^−/−) mouse model, we here report on their physiological role in lymphocyte differentiation and trafficking. CD18^−/− mice present with a defect in the distribution of lymphocytes with highly reduced numbers of naïve B and T lymphocytes in inguinal and axillary lymph nodes. In contrast, cervical lymph nodes were fourfold enlarged harbouring unconventional T-cell receptor-αβ (TCR-αβ) and TCR-γδ CD3⁺ CD4⁻ CD8⁻ (double-negative; DN) T cells that expanded in situ. Using adoptive transfer experiments, we found that these cells did not home to peripheral lymph nodes of CD18^wt recipients but, like antigen-experienced T or natural killer (NK) T cells, recirculated through non-lymphoid organs. Lacking regulatory functions in vitro, CD18^−/− TCR-αβ DN T cells did not suppress the proliferation of polyclonally activated CD4⁺ or CD8⁺ (single-positive; SP) T cells. Most interestingly, CD18^−/− TCR-αβ DN T cells showed intermediate TCR expression levels, an absent activation through allogeneic major histocompatibility complex and a strong proliferative dependence on interleukin-2, hence, closely resembling NKT cells. However, our data oppose former reports, clearly showing that, because of an absent reactivity with CD1d-αGalCer dimers, these cells are not mature classical NKT cells. Our data indicate that CD18^−/− TCR-αβ DN T cells, like NKT and TCR-γδ T cells, share characteristics of both adaptive and innate immune cells, and may accumulate as a compensatory mechanism to the functional defect of adaptive immunity in CD18^−/− mice.     

The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3^−/− mice had a higher mortality rate than C3^+/+ mice. Factor B^−/− mice, but not C4^−/− mice, infected with P. aeruginosa had a mortality rate similar to that of C3^−/− mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3^−/− mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3^+/+ mice at 24 h postinfection. In vitro, phagocytic cells from C3^+/+ or C3^−/− mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3^−/− mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1β (IL-1β), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3^+/+ mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.     

Type 1 interferon-induced IL-7 maintains CD8+ T-cell responses and homeostasis by suppressing PD-1 expression in viral hepatitis

Type 1 interferon (IFN-I) promotes antigen-presenting cell maturation and was recently shown to induce hepatic IL-7 production during infection. Herein, we further explored the underlying mechanisms used by IFN-I to orchestrate antiviral immune responses in the liver. Acute viral hepatitis was induced by i.v. injection of adenovirus (Ad) in IFN-α receptor knockout (IFNAR^−/−) and control mice. To disrupt signaling, monoclonal antibodies (mAbs) against IL-7 receptor alpha (IL-7Rα) or PD-L1 were i.p. injected. We found that CD8⁺ T cells in IFNAR^−/− mice were less effective than those in control mice. The reduced T-cell function was accompanied by increased levels of PD-1 expression, apoptosis and decreased IFN-γ production. The lack of IFN-I signaling also impaired the expression of accessory molecules in both intrahepatic dendritic cell (DCs) and hepatocytes. PD-L1 was comparably and highly expressed on hepatocytes in both IFNAR^−/− and control mice. Injection of PD-L1-specific mAb in IFNAR^−/− mice reversed the compromised immune responses in the liver. Further investigation showed that hepatic IL-7 elevation was less pronounced in IFNAR^−/− mice compared to the controls. A treatment with recombinant IL-7 suppressed PD-1 expression on CD8⁺ T cells in vitro. Accordingly, blocking IL-7R signaling in vivo resulted in increased PD-1 expression on CD8⁺ T cells in Ad-infected mice. Collectively, the results suggest that IFN-I-induced hepatic IL-7 production maintains antiviral CD8⁺ T-cell responses and homeostasis by suppressing PD-1 expression in acute viral hepatitis.     

Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4⁺ T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2^−/− mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2^−/− mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR^−/− nor RAG/IL-4Rα^−/− mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR^−/− mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα^−/− mice. RAG/IFN-γR^−/− mice had elevated numbers of lung CD4⁺ T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8⁺ T suppressor cells. Impaired lung CD8⁺ T cell responses in RAG/IFN-γR^−/− mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8⁺ T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.     

MZB1 is a GRP94 cochaperone that enables proper immunoglobulin heavy chain biosynthesis upon ER stress

MZB1 (pERp1) is a B-cell-specific and endoplasmic reticulum (ER)-localized protein implicated in antibody secretion and integrin-mediated cell adhesion. Here, we examine the role of MZB1 in vivo by conditional gene inactivation in the mouse germline and at different stages of B lymphopoiesis. Deletion of MZB1 impairs humoral immune responses and antibody secretion in plasma cells that naturally undergo ER stress. In addition, we found that experimental induction of ER stress by tunicamycin injections in mice results in a block of pro-B-cell to pre-B-cell differentiation specifically in Mzb1^−/− mice. A similar developmental block was observed in Mzb1^fl/flmb1^Cre mice, whereby a Cre recombinase-induced genotoxic stress unmasks a role for MZB1 in the surface expression of immunoglobulin µ heavy chains (µHCs). MZB1 associates directly with the substrate-specific chaperone GRP94 (also called HSP90B1 or gp96) in an ATP-sensitive manner and is required for the interaction of GRP94 with µHCs upon ER stress. Thus, MZB1 seems to act as a substrate-specific cochaperone of GRP94 that enables proper biosynthesis of µHCs under conditions of ER stress.     

Functional characterization of BRCA1 gene variants by mini-gene splicing assay

Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213−1G>A, c.670+1delG, c.4185+1G>A, and c.5075−1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302−15C>G, c.547+14delG, c.4676−20A>G, c.4987−21G>T, and c.5278−14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members.     

Toll-like receptor 7 deficiency suppresses type 1 diabetes development by modulating B-cell differentiation and function

Innate immunity mediated by Toll-like receptors (TLRs), which can recognize pathogen molecular patterns, plays a critical role in type 1 diabetes development. TLR7 is a pattern recognition receptor that senses single-stranded RNAs from viruses and host tissue cells; however, its role in type 1 diabetes development remains unclear. In our study, we discovered that Tlr7-deficient (Tlr7^−/−) nonobese diabetic (NOD) mice, a model of human type 1 diabetes, exhibited a significantly delayed onset and reduced incidence of type 1 diabetes compared with Tlr7-sufficient (Tlr7^+/+) NOD mice. Mechanistic investigations showed that Tlr7 deficiency significantly altered B-cell differentiation and immunoglobulin production. Moreover, Tlr7^−/− NOD B cells were found to suppress diabetogenic CD4⁺ T-cell responses and protect immunodeficient NOD mice from developing diabetes induced by diabetogenic T cells. In addition, we found that Tlr7 deficiency suppressed the antigen-presenting functions of B cells and inhibited cytotoxic CD8⁺ T-cell activation by downregulating the expression of both nonclassical and classical MHC class I (MHC-I) molecules on B cells. Our data suggest that TLR7 contributes to type 1 diabetes development by regulating B-cell functions and subsequent interactions with T cells. Therefore, therapeutically targeting TLR7 may prove beneficial for disease protection.     

The Granuloma Response Controlling Cryptococcosis in Mice Depends on the Sphingosine Kinase 1–Sphingosine 1-Phosphate Pathway

Cryptococcus neoformans is a fungal pathogen that causes pulmonary infections, which may progress into life-threatening meningitis. In commonly used mouse models of C. neoformans infections, fungal cells are not contained in the lungs, resulting in dissemination to the brain. We have previously reported the generation of an engineered C. neoformans strain (C. neoformans Δgcs1) which can be contained in lung granulomas in the mouse model and have shown that granuloma formation is dependent upon the enzyme sphingosine kinase 1 (SK1) and its product, sphingosine 1-phosphate (S1P). In this study, we have used four mouse models, CBA/J and C57BL6/J (both immunocompetent), Tgε26 (an isogenic strain of strain CBA/J lacking T and NK cells), and SK^−/− (an isogenic strain of strain C57BL6/J lacking SK1), to investigate how the granulomatous response and SK1-S1P pathway are interrelated during C. neoformans infections. S1P and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the bronchoalveolar lavage fluid of all mice infected with C. neoformans Δgcs1 but not in mice infected with the C. neoformans wild type. SK1^−/− mice did not show elevated levels of S1P or MCP-1. Primary neutrophils isolated from SK1^−/− mice showed impaired antifungal activity that could be restored by the addition of extracellular S1P. In addition, high levels of tumor necrosis factor alpha were found in the mice infected with C. neoformans Δgcs1 in comparison to the levels found in mice infected with the C. neoformans wild type, and their levels were also dependent on the SK1-S1P pathway. Taken together, these results suggest that the SK1-S1P pathway promotes host defense against C. neoformans infections by regulating cytokine levels, promoting extracellular killing by phagocytes, and generating a granulomatous response.     

Chlamydial Infection in Inducible Nitric Oxide Synthase Knockout Mice

Type 1 CD4⁺-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4⁺-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-γ)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS^+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS^−/− mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS^+/+ and iNOS^−/− mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS^−/− than in iNOS^+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS^−/− mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS^−/− mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-γ during CD4⁺-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-γ. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.     

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m² UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.     

Ras-Mek-Erk Signaling Regulates Nf1 Heterozygous Neointima Formation

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1^+/−) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1^+/− macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1^+/− neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1^+/− mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1^+/− neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes the protein neurofibromin. Neurofibromin negatively regulates Ras activity in multiple cell types by accelerating the hydrolysis of active Ras-GTP to its inactive diphosphate conformation.¹ These loss-of-function mutations accelerate Ras signaling and sensitize vessel wall cells and circulating hematopoietic cells, particularly myeloid progenitors and their differentiated progeny, to growth factors implicated in maintaining vascular wall homeostasis and disease pathogenesis.^1–4 Some patients with NF1 are predisposed to intimal proliferation, termed neointima, leading to debilitating arterial stenosis and tissue ischemia that contribute significantly to the premature mortality observed in this population.⁵Nf1 heterozygous (Nf1^+/−) mice display increased neointima formation, characterized by proliferating vascular smooth muscle cells (VSMCs) and infiltration of bone marrow–derived macrophages after arterial ligation, which is reminiscent of patients with NF1.^5,6 Neurofibromin-deficient endothelial cells, VSMCs, and bone marrow–derived myeloid cells demonstrate preferential activation of the Ras-Erk signaling pathway, without corresponding alterations in Ras–phosphatidylinositol 3-kinase signaling, in response to multiple growth factors in vitro.^2–4,7 This is an interesting observation because lineage-restricted inactivation of a single Nf1 gene in endothelial cells and/or VSMCs does not replicate the striking neointima observed in Nf1 heterozygous mice. However, we recently demonstrated that lineage-specific inactivation of a single Nf1 gene copy in monocytes/macrophages is sufficient to reproduce the enhanced neointima formation observed in Nf1 heterozygous mice compared with wild-type (WT) mice.⁸Based on these observations, we used in vitro and in vivo systems of macrophage function to test the hypothesis that Nf1 heterozygous macrophage function and mobilization to sites of inflammation are directly controlled by Ras-Erk signaling and that use of a specific and long-acting inhibitor of Ras-Erk signaling, under evaluation in multiple phase 1 and 2 clinical trials for cancer and preclinical models of NF1 malignancy,^1,9–12 will reduce neointima formation after mechanical injury.