Chronic obstructive pulmonary disease (COPD) is a progressive condition characterized by chronic airway inflammation and airspace remodeling, leading to airflow limitation that is not completely reversible. Smoking is the leading risk factor for compromised lung function stemming from COPD pathogenesis. First- and second-hand cigarette smoke contain thousands of constituents, including several carcinogens and cytotoxic chemicals that orchestrate chronic lung inflammation and destructive alveolar remodeling. Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors primarily expressed by diverse lung cells. RAGE expression increases following cigarette smoke exposure and expression is elevated in the lungs of patients with COPD. RAGE is responsible in part for inducing pro-inflammatory signaling pathways that culminate in expression and secretion of several cytokines, chemokines, enzymes, and other mediators. In the current review, new transgenic mouse models that conditionally over-express RAGE in pulmonary epithelium are discussed. When RAGE is over-expressed throughout embryogenesis, apoptosis in the peripheral lung causes severe lung hypoplasia. Interestingly, apoptosis in RAGE transgenic mice occurs via conserved apoptotic pathways also known to function in advanced stages of COPD. RAGE over-expression in the adult lung models features of COPD including pronounced inflammation and loss of parenchymal tissue. Understanding the biological contributions of RAGE during cigarette smoke-induced inflammation may provide critically important insight into the pathology of COPD.

Receptors for advanced glycation end-products (RAGE) are multiligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages, and expression increases following exposure to cigarette smoke extract (CSE). The present study sought to characterize the proinflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE knockout (KO) mice compared with controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and analyzed. CSE significantly increased RAGE expression by wild-type AMs. Employing ELISAs, wild-type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates proinflammatory signaling. Conversely, RAGE KO AMs had less Ras activation compared with wild-type AMs after exposure to CSE. In RAGE KO AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed that CSE-induced inflammation may occur in part via RAGE signaling. Lastly, quantitative RT-PCR revealed that the expression of proinflammatory cytokines including TNF-α and IL-1β were detectably decreased in RAGE KO AMs exposed to CSE compared with CSE-exposed wild-type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.

Receptors for advanced glycation end-products (RAGEs) are multiligand cell surface receptors highly expressed in the lung that contribute to alveolar epithelial cell differentiation during embryogenesis and the modulation of pulmonary inflammation during disease. When RAGEs are overexpressed throughout embryogenesis, severe lung hypoplasia ensues, culminating in perinatal lethality. However, the possible mechanisms that lead to the disappearance of pulmonary tissue remain unclear. A time course of lung organogenesis, commencing on Embryonic Day (E) 12.5, demonstrated that increased RAGE expression primarily alters lung morphogenesis beginning on E16.5. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunohistochemistry and immunoblotting for active caspase-3 confirmed a shift toward apoptosis in lungs from RAGE-overexpressing mice, compared with wild-type control mice. This observation supports previous work where electron microscopy identified the cellular blebbing of alveolar epithelium in embryonic RAGE-overexpressing mice. Assaying for NF-κB also revealed elevated nuclear translocation in lungs from transgenic mice compared with control mice. An RT-PCR assessment of genes regulated by NF-κB demonstrated the elevated expression of Fas ligand, suggesting increased activity of the Fas-mediated signal transduction pathway in which ligand-receptor interactions trigger cell death. These data provide evidence that the expression of RAGEs must be tightly regulated during homeostatic organogenesis. Further elucidations of the RAGE signaling potentially involved in cell-cycle abnormalities may provide insights into the progression of RAGE-mediated lung diseases.

Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-κB in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNFα, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.

BackgroundReceptors for advanced glycation end-products (RAGE) are cell surface receptors prominently expressed by lung epithelium. Previous research demonstrated that over-expression of RAGE by murine alveolar epithelial cells during embryogenesis caused severe lung hypoplasia and neonatal lethality. However, the effects of RAGE over-expression on adjacent matrix and endothelial cells remained unknown.MethodsRAGE transgenic (TG) mice were generated that conditionally over-expressed RAGE in alveolar type II cells when fed doxycycline (dox) from conception to E18.5. To evaluate effects on the basement membrane, immunostaining and immunoblotting were performed for collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. To assess changes in vasculature, immunostaining, immunoblotting and qRT-PCR were performed for Pecam-1, a platelet endothelial cell adhesion marker also known as CD31. Lastly, to characterize potential regulatory mechanisms of endothelial cell differentiation, immunoblotting and qRT-PCR for FoxM1, a key endothelium-specific transcription factor of the Forkhead Box (Fox) family, were completed.ResultsQualitative immunostaining for collagen IV was less in RAGE TG mice compared to controls and immunoblotting revealed decreased collagen IV in the RAGE TG mouse lung. Additionally, elevated MMP-9 detected via immunostaining and immunoblotting implicated MMP-9 as a possible down stream effector in matrix destabilization mediated by RAGE signaling. Lastly, Pecam-1 assessment revealed a decrease in the prevalence of microvascular endothelial cells coincident with FoxM1 abrogation in RAGE TG mice compared to controls.ConclusionsRAGE over-expression by alveolar epithelium weakened the basement membrane and associated matrix via increased MMP-9 activity. Furthermore, over-expression of RAGE inhibited FoxM1, suggesting that anomalous transcriptional control contributes to decreased endothelial cell prevalence in the TG mouse lung.

Triple-negative breast cancer (TNBC) represents 15% of all breast carcinomas. The unfavorable prognosis and aggressive biology of TNBC highlight the need to find new targeted therapies. Previous study from our lab has shown that the prolyl hydroxylase EglN2 contributes to TNBC progression, with its depletion leading to a decrease in TNBC cell invasion. However, the underlying mechanism remains elusive. In our current study, we developed a novel enzyme-substrate trapping strategy followed by mass spectrometry and identified adenylosuccinate lyase (ADSL) as a potential EglN2 hydroxylase substrate. EglN2 binds directly with ADSL and catalyzes ADSL hydroxylation on proline residues, which might affect ADSL activity. We demonstrated that ADSL is specifically upregulated in TNBC cell lines and patients. Functionally, depletion of ADSL led to decreased anchorage-independent growth, TNBC cell invasion, and orthotopic tumor growth. Mechanistically, integrated analyses of metabolomics and RNA-seq showed that ADSL controls pyrimidine biosynthesis that is essential for TNBC cell proliferation and invasion. Therefore, our study suggests that EglN2-ADSL signaling axis can serve as a novel therapeutic avenue in TNBC by regulating pyrimidine metabolism. Citation Format: Giada Zurlo, Jeremy Simon, Cheng Fan, Adam Robinson, Javier Rodriguez Martinez, Alex Kriegsheim, Jason Locasale, Charles Perou, Qing Zhang. ADSL controls pyrimidine metabolism and triple-negative breast tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B18.

Receptors for advanced glycation end‐products (RAGE) are members of the immunoglobulin superfamily of cell‐surface receptors implicated in pulmonary inflammation. In the present study, we tested the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) triggered by diesel particulate matter (DPM). Using real time RT‐PCR, we discovered that RAGE was up‐regulated in primary AMs exposed to DPM for four hours. Because DPM increased RAGE expression, we exposed primary AMs isolated from RAGE null and wild type Balb/C mice to DPM. DPM led to the activation p38 and NF‐kB and that these two signaling intermediates were diminished in similarly exposed AMs isolated from RAGE null mice. Furthermore, TNF‐a, IL‐12, and IL‐13 were all significantly decreased in culture media from DPM‐exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that primary AMs orchestrate diesel‐induced inflammation, at least in part, via RAGE‐mediated mechanisms. Further work may show that RAGE signaling is a potential target in treatment of inflammatory lung diseases exacerbated by environmental pollution. This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).

Aberrant expression of the epithelial cell adhesion protein P-cadherin has been observed to be independently causative and preventative with respect to tumor progression, depending on tissue type. Oral epithelial tumors have been shown to exhibit a unique signature of P-cadherin expression in which P-cadherin expression increases in dysplasia and is subsequently lost during malignancy. These studies suggest that early increases in P-cadherin expression may be critical events in tumor progression. Since E- and N-cadherin have been shown to physically interact with and modulate function of Epidermal Growth Factor (EGF) and Fibroblast Growth Factor (FGF) respectively, we hypothesized that P-cadherin could promote tumor development via its own interaction with receptor tyrosine kinases in a ligand-dependent manner. To identify functional interactions between P-cadherin and receptor tyrosine kinases, we retrovirally transduced two independently isolated oral squamous carcinoma cell lines to overexpress P-cadherin. Transduced cells were treated independently with one of seven different growth factors to identify alterations in mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling pathways. For all experiments, protein was collected over the course of six hours at multiple timepoints to identify fluctuations in signaling. Although P-cadherin did not alter AKT phosphorylation, it was found to alter MAPK signaling in both a positive and negative manner. The most robust alterations in MAPK signaling occurred in response to activation of Insulin-like Growth Factor (IGF), Epithelial Growth Factor (EGF), and Keratinocyte Growth Factor (KGF). P-cadherin overexpression also induced phenotypic changes. Behavioral assays revealed that P-cadherin expression increased motility, but decreased expression of invasive proteases MMP-9 and MMP-14. P-cadherin cells grown in serum-free media for 48 hours exhibited increased cell number compared to control cells. A cell proliferation analysis (MTT assay) indicated lower rates of proliferation in P-cadherin -overexpressing cells compared to controls. This data suggests that the increased cell number of serum-starved P-cadherin cells was due to increased survival, rather than proliferation. These results demonstrate that P-cadherin overexpression can modulate ligand-dependent growth factor signaling and alter those cellular characteristics most commonly associated with tumor aggressiveness. Since P-cadherin expression has been shown to increase early on in the development of tumors, our research may provide a possible mechanism as to how this increase may play a role in tumor progression during dysplasia. Citation Format: James Mar, Adam Robinson, Rachel Neville, Desseree Lysne, Kathryn Lawson. P-cadherin modulates signaling of multiple growth factor receptors and cellular aggressiveness in oral carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4409. doi:10.1158/1538-7445.AM2014-4409