TLR7 is expressed by support cells, but not sensory neurons, in ganglia.

BACKGROUND: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. METHODS: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. RESULTS: In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. CONCLUSION: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7's neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7's primary role in neuronal tissues is not related to antiviral immunity.

Chronic cerebral lipocalin 2 exposure elicits hippocampal neuronal dysfunction and cognitive impairment.

Lipocalin 2 (LCN2) is a pleiotropic molecule that is induced in the central nervous system (CNS) in several acute and chronic pathologies. The acute induction of LCN2 evolved as a beneficial process, aimed at combating bacterial infection through the sequestration of iron from pathogens, while the role of LCN2 during chronic, non-infectious disease remains unclear, and recent studies suggest that LCN2 is neurotoxic. However, whether LCN2 is sufficient to induce behavioral and cognitive alterations remains unclear. In this paper, we sought to address the role of cerebral LCN2 on cognition in both acute and chronic settings. We demonstrate that LCN2 is robustly induced in the CNS during both acute and chronic inflammatory conditions, including LPS-based sepsis and cancer cachexia. In vivo, LPS challenge results in a global induction of LCN2 in the central nervous system, while cancer cachexia results in a distribution specific to the vasculature. Similar to these in vivo observations, in vitro modeling demonstrated that both glia and cerebral endothelium produce and secrete LCN2 when challenged with LPS, while only cerebral endothelium secrete LCN2 when challenged with cancer-conditioned medium. Chronic, but not short-term, cerebral LCN2 exposure resulted in reduced hippocampal neuron staining intensity, an increase in newborn neurons, microglial activation, and increased CNS immune cell infiltration, while gene set analyses suggested these effects were mediated through melanocortin-4 receptor independent mechanisms. RNA sequencing analyses of primary hippocampal neurons revealed a distinct transcriptome associated with prolonged LCN2 exposure, and ontology analysis was suggestive of altered neurite growth and abnormal spatial learning. Indeed, LCN2-treated hippocampal neurons display blunted neurite processes, and mice exposed to prolonged cerebral LCN2 levels experienced a reduction in spatial reference memory as indicated by Y-maze assessment. These findings implicate LCN2 as a pathologic mediator of cognitive decline in the setting of chronic disease.

Lipocalin 2 mediates appetite suppression during pancreatic cancer cachexia.

Lipocalin 2 (LCN2) was recently identified as an endogenous ligand of the type 4 melanocortin receptor (MC4R), a critical regulator of appetite. However, it remains unknown if this molecule influences appetite during cancer cachexia, a devastating clinical entity characterized by decreased nutrition and progressive wasting. We demonstrate that LCN2 is robustly upregulated in murine models of pancreatic cancer, its expression is associated with reduced food consumption, and Lcn2 deletion is protective from cachexia-anorexia. Consistent with LCN2's proposed MC4R-dependent role in cancer-induced anorexia, pharmacologic MC4R antagonism mitigates cachexia-anorexia, while restoration of Lcn2 expression in the bone marrow is sufficient in restoring the anorexia feature of cachexia. Finally, we observe that LCN2 levels correlate with fat and lean mass wasting and is associated with increased mortality in patients with pancreatic cancer. Taken together, these findings implicate LCN2 as a pathologic mediator of appetite suppression during pancreatic cancer cachexia.

Circulating myeloid cells invade the central nervous system to mediate cachexia during pancreatic cancer.

Weight loss and anorexia are common symptoms in cancer patients that occur prior to initiation of cancer therapy. Inflammation in the brain is a driver of these symptoms, yet cellular sources of neuroinflammation during malignancy are unknown. In a mouse model of pancreatic ductal adenocarcinoma (PDAC), we observed early and robust myeloid cell infiltration into the brain. Infiltrating immune cells were predominately neutrophils, which accumulated at a unique central nervous system entry portal called the velum interpositum, where they expressed CCR2. Pharmacologic CCR2 blockade and genetic deletion of Ccr2 both resulted in significantly decreased brain-infiltrating myeloid cells as well as attenuated cachexia during PDAC. Lastly, intracerebroventricular blockade of the purinergic receptor P2RX7 during PDAC abolished immune cell recruitment to the brain and attenuated anorexia. Our data demonstrate a novel function for the CCR2/CCL2 axis in recruiting neutrophils to the brain, which drives anorexia and muscle catabolism.

Weight loss, decreased appetite and fatigue are symptoms of a wasting disorder known as cachexia, which is common in several serious diseases such as AIDS, chronic lung disease and heart failure. Up to 80 percent of people with advanced cancer also develop cachexia, and there are no effective treatments. It is not known how cachexia develops, but symptoms like appetite loss and fatigue are controlled by the brain. One theory is that the brain may be responding to a malfunctioning immune response that causes inflammation. While the brain was thought to be protected from this, new research has shown that it is possible for cells from the immune system to reach the brain in some conditions. To find out if this also happens in cancer, Burfeind et al. studied mice that had been implanted with pancreatic cancer cells and were showing signs of cachexia. Samples from the mice's brains showed that immune cells known as neutrophils were present and active. A protein known as CCR2 was found in higher levels in the brains of these mice. This protein is involved in the movement of neutrophil cells through the body. To see what effect this protein had, Burfeind et al. gave the mice a drug that blocks CCR2. This prevented the neutrophils from entering the brain and reduced the symptoms of cachexia in the mice. To further confirm the role of CCR2, the mice were genetically modified so that they could not produce the protein. This reduced the number of neutrophils seen in the brain but not in the rest of the body. This suggests that a drug targeting CCR2 could help to reduce the symptoms of cachexia, without disrupting the normal immune response away from the brain. This approach would still need to be tested in clinical trials before it is possible to know how effective it might be in humans.

Microglia in the hypothalamus respond to tumor-derived factors and are protective against cachexia during pancreatic cancer.

Microglia in the mediobasal hypothalamus (MBH) respond to inflammatory stimuli and metabolic perturbations to mediate body composition. This concept is well studied in the context of high fat diet induced obesity (HFDO), yet has not been investigated in the context of cachexia, a devastating metabolic syndrome characterized by anorexia, fatigue, and muscle catabolism. We show that microglia accumulate specifically in the MBH early in pancreatic ductal adenocarcinoma (PDAC)-associated cachexia and assume an activated morphology. Furthermore, we observe astrogliosis in the MBH and hippocampus concurrent with cachexia initiation. We next show that circulating immune cells resembling macrophages infiltrate the MBH. PDAC-derived factors induced microglia to express a transcriptional profile in vitro that was distinct from that induced by lipopolysaccharide (LPS). Microglia depletion through CSF1-R antagonism resulted in accelerated cachexia onset and increased anorexia, fatigue, and muscle catabolism during PDAC. This corresponded with increased hypothalamic-pituitary-adrenal (HPA) axis activation. CSF1-R antagonism had little effect on inflammatory response in the circulation, liver, or tumor. These findings demonstrate that microglia are protective against PDAC cachexia and provide mechanistic insight into this function.

Publisher Correction: The TLR7/8 agonist R848 remodels tumor and host responses to promote survival in pancreatic cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The TLR7/8 agonist R848 remodels tumor and host responses to promote survival in pancreatic cancer.

A priority in cancer research is to innovate therapies that are not only effective against tumor progression but also address comorbidities such as cachexia that limit quality and quantity of life. We demonstrate that TLR7/8 agonist R848 induces anti-tumor responses and attenuates cachexia in murine models of pancreatic ductal adenocarcinoma (PDAC). In vivo, tumors from two of three cell lines were R848-sensitive, resulting in smaller tumor mass, increased immune complexity, increased CD8+ T-cell infiltration and activity, and decreased Treg frequency. R848-treated mice demonstrated improvements in behavioral and molecular cachexia manifestations, resulting in a near-doubling of survival duration. Knockout mouse studies revealed that stromal, not neoplastic, TLR7 is requisite for R848-mediated responses. In patient samples, we found Tlr7 is ubiquitously expressed in stroma across all stages of pancreatic neoplasia, but epithelial Tlr7 expression is relatively uncommon. These studies indicate immune-enhancing approaches including R848 may be useful in PDAC and cancer-associated cachexia.

Persistent Toll-like receptor 7 stimulation induces behavioral and molecular innate immune tolerance.

Toll-like receptors 7 and 8 (TLR7 and TLR8) are endosomal pattern recognition receptors that detect a variety of single-stranded RNA species. While TLR7/8 agonists have robust therapeutic potential, clinical utility of these agents is limited by sickness responses associated with treatment induction. To understand the kinetics and mechanism of these responses, we characterized the acute and chronic effects of TLR7 stimulation. Single-cell RNA-sequencing studies, RNAscope, and radiolabeled in situ hybridization demonstrate that central nervous system gene expression of TLR7 is exclusive to microglia. In vitro studies demonstrate that microglia are highly sensitive to TLR7 stimulation, and respond in a dose-dependent manner to the imidazoquinoline R848. In vivo, both intraperitoneal (IP) and intracerebroventricular (ICV) R848 induce acute sickness responses including hypophagia, weight loss, and decreased voluntary locomotor activity, associated with increased CNS pro-inflammatory gene expression and changes to glial morphology. However, chronic daily IP R848 resulted in rapid tachyphylaxis of behavioral and molecular manifestations of illness. In microglial in vitro assays, pro-inflammatory transcriptional responses rapidly diminished in the context of repeated R848. In addition to TLR7 desensitization, we found that microglia become partially refractory to lipopolysaccharide (LPS) following R848 pretreatment, associated with induction of negative regulators A20 and Irak3. Similarly, mice pre-treated with R848 demonstrate reduced sickness responses, hypothalamic inflammation, and hepatic inflammation in response to LPS. These data combined demonstrate that TLR7 stimulation induces acute behavioral and molecular evidence of sickness responses. Following prolonged dosing, R848 induces a refractory state to both TLR7 and TLR4 activation, consistent with induced immune tolerance.

MyD88 signalling is critical in the development of pancreatic cancer cachexia.

BACKGROUND: Up to 80% of pancreatic cancer patients suffer from cachexia, a devastating condition that exacerbates underlying disease, reduces quality of life, and increases treatment complications and mortality. Tumour-induced inflammation is linked to this multifactorial wasting syndrome, but mechanisms and effective treatments remain elusive. Myeloid differentiation factor (MyD88), a key component of the innate immune system, plays a pivotal role in directing the inflammatory response to various insults. In this study, we tested whether MyD88 signalling is essential in the development of pancreatic cancer cachexia using a robust mouse tumour model. METHODS: Sex, age, and body weight-matched wide type (WT) and MyD88 knockout (MyD88 KO) mice were orthotopically or intraperitoneally implanted with a pancreatic tumour cell line from a syngeneic C57BL/6 KRASG12D/+ P53R172H/+ Pdx-Cre (KPC) mouse. We observed the effects of MyD88 signalling during pancreatic ductal adenocarcinoma progression and the cachexia development through behavioural, histological, molecular, and survival aspects. RESULTS: Blocking MyD88 signalling greatly ameliorated pancreatic ductal adenocarcinoma-associated anorexia and fatigue, attenuated lean mass loss, reduced muscle catabolism and atrophy, diminished systemic and central nervous system inflammation, and ultimately improved survival. Our data demonstrate that MyD88 signalling plays a critical role in mediating pancreatic cancer-induced inflammation that triggers cachexia development and therefore represents a promising therapeutic target. CONCLUSIONS: MyD88-dependent inflammation is crucial in the pathophysiology of pancreatic cancer progression and contributes to high mortality. Our findings implicate the importance of innate immune signalling pathways in pancreatic cancer cachexia and a novel therapeutic target.

TRIF is a key inflammatory mediator of acute sickness behavior and cancer cachexia.

Hypothalamic inflammation is a key component of acute sickness behavior and cachexia, yet mechanisms of inflammatory signaling in the central nervous system remain unclear. Previous work from our lab and others showed that while MyD88 is an important inflammatory signaling pathway for sickness behavior, MyD88 knockout (MyD88KO) mice still experience sickness behavior after inflammatory stimuli challenge. We found that after systemic lipopolysaccharide (LPS) challenge, MyD88KO mice showed elevated expression of several cytokine and chemokine genes in the hypothalamus. We therefore assessed the role of an additional inflammatory signaling pathway, TRIF, in acute inflammation (LPS challenge) and in a chronic inflammatory state (cancer cachexia). TRIFKO mice resisted anorexia and weight loss after peripheral (intraperitoneal, IP) or central (intracerebroventricular, ICV) LPS challenge and in a model of pancreatic cancer cachexia. Compared to WT mice, TRIFKO mice showed attenuated upregulation of Il6, Ccl2, Ccl5, Cxcl1, Cxcl2, and Cxcl10 in the hypothalamus after IP LPS treatment, as well as attenuated microglial activation and neutrophil infiltration into the brain after ICV LPS treatment. Lastly, we found that TRIF was required for Ccl2 upregulation in the hypothalamus and induction of the catabolic genes, Mafbx, Murf1, and Foxo1 in gastrocnemius during pancreatic cancer. In summary, our results show that TRIF is an important inflammatory signaling mediator of sickness behavior and cachexia and presents a novel therapeutic target for these conditions.

Establishment and characterization of a novel murine model of pancreatic cancer cachexia.

BACKGROUND: Cachexia is a complex metabolic and behavioural syndrome lacking effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is one of the most important conditions associated with cachexia, with >80% of PDAC patients suffering from the condition. To establish the cardinal features of a murine model of PDAC-associated cachexia, we characterized the effects of implanting a pancreatic tumour cell line from a syngeneic C57BL/6 KRASG12D P53R172H Pdx-Cre+/+ (KPC) mouse. METHODS: Male and female C57BL/6 mice were inoculated subcutaneously, intraperitoneally, or orthotopically with KPC tumour cells. We performed rigorous phenotypic, metabolic, and behavioural analysis of animals over the course of tumour development. RESULTS: All routes of administration produced rapidly growing tumours histologically consistent with moderate to poorly differentiated PDAC. The phenotype of this model was dependent on route of administration, with orthotopic and intraperitoneal implantation inducing more severe cachexia than subcutaneous implantation. KPC tumour growth decreased food intake, decreased adiposity and lean body mass, and decreased locomotor activity. Muscle catabolism was observed in both skeletal and cardiac muscles, but the dominant catabolic pathway differed between these tissues. The wasting syndrome in this model was accompanied by hypothalamic inflammation, progressively decreasing brown and white adipose tissue uncoupling protein 1 (Ucp1) expression, and increased peripheral inflammation. Haematological and endocrine abnormalities included neutrophil-dominant leukocytosis and anaemia, and decreased serum testosterone. CONCLUSIONS: Syngeneic KPC allografts are a robust model for studying cachexia, which recapitulate key features of the PDAC disease process and induce a wide array of cachexia manifestations. This model is therefore ideally suited for future studies exploring the physiological systems involved in cachexia and for preclinical studies of novel therapies.

A distinct brain pathway links viral RNA exposure to sickness behavior.

Sickness behaviors and metabolic responses to invading pathogens are common to nearly all types of infection. These responses evolved to provide short-term benefit to the host to ward off infection, but impact on quality of life, and when prolonged lead to neurodegeneration, depression, and cachexia. Among the major infectious agents, viruses most frequently enter the brain, resulting in profound neuroinflammation. We sought to define the unique features of the inflammatory response in the brain to these infections. We demonstrate that the molecular pathway defining the central response to dsRNA is distinct from that found in the periphery. The behavioral and physical response to the dsRNA mimetic poly I:C is dependent on signaling via MyD88 when it is delivered centrally, whereas this response is mediated via the TRIF pathway when delivered peripherally. We also define the likely cellular candidates for this MyD88-dependent step. These findings suggest that symptom management is possible without ameliorating protective antiviral immune responses.

The central role of hypothalamic inflammation in the acute illness response and cachexia.

When challenged with a variety of inflammatory threats, multiple systems across the body undergo physiological responses to promote defense and survival. The constellation of fever, anorexia, and fatigue is known as the acute illness response, and represents an adaptive behavioral and physiological reaction to stimuli such as infection. On the other end of the spectrum, cachexia is a deadly and clinically challenging syndrome involving anorexia, fatigue, and muscle wasting. Both of these processes are governed by inflammatory mediators including cytokines, chemokines, and immune cells. Though the effects of cachexia can be partially explained by direct effects of disease processes on wasting tissues, a growing body of evidence shows the central nervous system (CNS) also plays an essential mechanistic role in cachexia. In the context of inflammatory stress, the hypothalamus integrates signals from peripheral systems, which it translates into neuroendocrine perturbations, altered neuronal signaling, and global metabolic derangements. Therefore, we will discuss how hypothalamic inflammation is an essential driver of both the acute illness response and cachexia, and why this organ is uniquely equipped to generate and maintain chronic inflammation. First, we will focus on the role of the hypothalamus in acute responses to dietary and infectious stimuli. Next, we will discuss the role of cytokines in driving homeostatic disequilibrium, resulting in muscle wasting, anorexia, and weight loss. Finally, we will address mechanisms and mediators of chronic hypothalamic inflammation, including endothelial cells, chemokines, and peripheral leukocytes.

Sustained hyperoxia-induced NF-κB activation improves survival and preserves lung development in neonatal mice.

Oxygen toxicity contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Neonatal mice exposed to hyperoxia develop a simplified lung structure that resembles BPD. Sustained activation of the transcription factor NF-κB and increased expression of protective target genes attenuate hyperoxia-induced mortality in adults. However, the effect of enhancing hyperoxia-induced NF-κB activity on lung injury and development in neonatal animals is unknown. We performed this study to determine whether sustained NF-κB activation, mediated through IκBß overexpression, preserves lung development in neonatal animals exposed to hyperoxia. Newborn wild-type (WT) and IκBß-overexpressing (AKBI) mice were exposed to hyperoxia (>95%) or room air from day of life (DOL) 0-14, after which all animals were kept in room air. Survival curves were generated through DOL 14. Lung development was assessed using radial alveolar count (RAC) and mean linear intercept (MLI) at DOL 3 and 28 and pulmonary vessel density at DOL 28. Lung tissue was collected, and NF-κB activity was assessed using Western blot for IκB degradation and NF-κB nuclear translocation. WT mice demonstrated 80% mortality through 14 days of exposure. In contrast, AKBI mice demonstrated 60% survival. Decreased RAC, increased MLI, and pulmonary vessel density caused by hyperoxia in WT mice were significantly attenuated in AKBI mice. These findings were associated with early and sustained NF-κB activation and expression of cytoprotective target genes, including vascular endothelial growth factor receptor 2. We conclude that sustained hyperoxia-induced NF-κB activation improves neonatal survival and preserves lung development. Potentiating early NF-κB activity after hyperoxic exposure may represent a therapeutic intervention to prevent BPD.

IκBß-mediated NF-κB activation confers protection against hyperoxic lung injury.

Supplemental oxygen is frequently used in an attempt to improve oxygen delivery; however, prolonged exposure results in damage to the pulmonary endothelium and epithelium. Although NF-κB has been identified as a redox-responsive transcription factor, whether NF-κB activation exacerbates or attenuates hyperoxic lung injury is unclear. We determined that sustained NF-κB activity mediated by IκBß attenuates lung injury and prevents mortality in adult mice exposed to greater than 95% O2. Adult wild-type mice demonstrated evidence of alveolar protein leak and 100% mortality by 6 days of hyperoxic exposure, and showed NF-κB nuclear translocation that terminated after 48 hours. Furthermore, these mice showed increased expression of NF-κB-regulated proinflammatory and proapoptotic cytokines. In contrast, mice overexpressing the NF-κB inhibitory protein, IκBß (AKBI), demonstrated significant resistance to hyperoxic lung injury, with 50% surviving through 8 days of exposure. This was associated with NF-κB nuclear translocation that persisted through 96 hours of exposure. Although induction of NF-κB-regulated proinflammatory cytokines was not different between wild-type and AKBI mice, significant up-regulation of antiapoptotic proteins (BCL-2, BCL-XL) was found exclusively in AKBI mice. We conclude that sustained NF-κB activity mediated by IκBß protects against hyperoxic lung injury through increased expression of antiapoptotic genes.

The NF-κB inhibitory proteins IκBα and IκBß mediate disparate responses to inflammation in fetal pulmonary endothelial cells.

Exposure to intrauterine inflammation impairs lung growth but paradoxically protects the neonatal pulmonary vasculature from hyperoxic injury. The mechanisms mediating these contradictory effects are unknown. The objective is to identify the role of NF-κB in mediating cytoprotective and proinflammatory responses to inflammation in the fetal pulmonary endothelium. In newborn rats exposed to intra-amniotic LPS, we found increased expression of the NF-κB target gene manganese superoxide dismutase (MnSOD) in the pulmonary endothelium. Supporting these in vivo findings, LPS induced NF-κB activation and MnSOD expression in isolated fetal pulmonary arterial endothelial cells. In addition, LPS exposure caused apoptosis and suppressed cellular growth and induced P-selectin expression. LPS-induced NF-κB activation that proceeded through specific isoforms of the inhibitory protein IκB mediated these diverse responses; NF-κB signaling through IκBα degradation resulted in MnSOD upregulation and preserved cell growth, whereas NF-κB signaling through IκBß degradation mediated apoptosis and P-selectin expression. These findings suggest that selective inhibition of NF-κB activation that results from IκBß degradation preserves the enhanced antioxidant defense and protects the developing pulmonary vascular endothelium from ongoing inflammatory injury.

Reprimo (RPRM) is a novel tumor suppressor in pituitary tumors and regulates survival, proliferation, and tumorigenicity.

Reprimo (RPRM), initially identified as a downstream effector of p53-induced cell cycle arrest at G(2)/M, is a putative tumor suppressor silenced in some types of cancer. In microarrays, the RPRM transcript was repressed 26-fold in gonadotrope (null cell) human pituitary tumors compared with normal pituitary but in the absence of changes in p53. Inhibition of RPRM mRNA was confirmed by RT-PCR in all gonadotrope tumors, most GH samples, and variably in other tumor types. Human pituitary tumors showed no evidence of abnormal promoter hypermethylation as a mechanism of RPRM repression. RPRM stable expression in gonadotrope (LßT2) and GH (GH3) pituitary cells resulted in decreased rates of cell proliferation by 55 and 30%, respectively; however, RPRM reexpression did not alter G(2)/M transition. In addition, RPRM increased rates of apoptosis in response to growth factor deprivation as assessed by caspase-3 cleavage and nuclear condensation. Clonagenic assays showed a 5.3- and 3.7-fold suppression of colony growth in RPRM-overexpressing LßT2 and GH3 cells, respectively, supporting its role as a tumor suppressor. In cells stably expressing RPRM mRNA, protein levels were actively suppressed due to rapid degradation through ubiquitination and proteasomal targeting. Growth factor withdrawal, as a model of cellular stress, stabilized RPRM protein levels. Together these data suggest that RPRM is transiently up-regulated at a posttranscriptional level in times of cellular stress to restrict cell survival, proliferation, and tumor formation. When RPRM is silenced as in human pituitary tumors, unrestrained growth and tumor progression may occur.

Nuclear factor-κB (NF-κB) inhibitory protein IκBß determines apoptotic cell death following exposure to oxidative stress.

The transcription factor NF-κB regulates the cellular response to inflammatory and oxidant stress. Although many studies have evaluated NF-κB activity following exposure to oxidative stress, the role of the IκB family of inhibitory proteins in modulating this activity remains unclear. Specifically, the function of IκBß in mediating the cellular response to oxidative stress has not been evaluated. We hypothesized that blocking oxidative stress-induced NF-κB signaling through IκBß would prevent apoptotic cell death. Using IκBß knock-in mice (AKBI), in which the IκBα gene is replaced with the IκBß cDNA, we show that IκBß overexpression prevented oxidative stress-induced apoptotic cell death. This was associated with retention of NF-κB subunits in the nucleus and maintenance of NF-κB activity. Furthermore, the up-regulation of pro-apoptotic genes in WT murine embryonic fibroblasts (MEFs) exposed to serum starvation was abrogated in AKBI MEFs. Inhibition of apoptosis was observed in WT MEFs overexpressing IκBß with simultaneous IκBα knockdown, whereas IκBß overexpression alone did not produce this effect. These findings represent a necessary but not sufficient role of IκBß in preventing oxidant stress-induced cell death.

Identification of growth arrest and DNA-damage-inducible gene beta (GADD45beta) as a novel tumor suppressor in pituitary gonadotrope tumors.

Gonadotrope and null cell pituitary tumors cause significant morbidity, often presenting with signs of hypogonadism together with visual disturbances due to mass effects. Surgery and radiation are the only therapeutic options to date. To identify dysregulated genes and pathways that may play a role in tumorigenesis and/or progression, molecular profiling was performed on 14 gonadotrope tumors, with nine normal human pituitaries obtained at autopsy serving as controls. Bioinformatic analysis identified putative downstream effectors of tumor protein 53 (p53) that were consistently repressed in gonadotrope pituitary tumors, including RPRM, P21, and PMAIP1, with concomitant inhibition of the upstream p53 regulator, PLAGL1(Zac1). Further analysis of the growth arrest and DNA damage-inducible (GADD45) family revealed no change in the p53 target, GADD45α, but identified repression of GADD45ß in pituitary tumors in addition to the previously reported inhibition of GADD45γ. Overexpression of GADD45ß in LßT2 mouse gonadotrope cells blocked tumor cell proliferation and increased rates of apoptosis in response to growth factor withdrawal. Stable gonadotrope cell transfectants expressing increased GADD45ß showed decreased colony formation in soft agar, confirming its normal role as a tumor suppressor. Unlike previous studies of GADD45γ in pituitary tumors and α and ß in other tumors, bisulfite sequencing showed no evidence of hypermethylation of the GADD45ß promoter in human pituitary tumor samples to explain the repression of its expression. Thus, GADD45ß is a novel pituitary tumor suppressor whose reexpression blocks proliferation, survival, and tumorigenesis. Together these studies identify new targets and mechanisms to explore in pituitary tumor initiation and progression.