Petra Isabel Lorenzo

Immunohistochemical assessment of Pax8 expression during pancreatic islet development and in human neuroendocrine tumors

The paired box transcription factor Pax8 is critical for development of the eye, thyroid gland as well as the urinary and reproductive organs. In adult, Pax8 overexpression is associated with kidney, ovarian and thyroid tumors and has emerged as a specific marker for these cancers. Recently, Pax8 expression was also reported in human pancreatic islets and in neuroendocrine tumors, identifying Pax8 as a novel member of the Pax family expressed in the pancreas. Herein, we sought to provide a comprehensive analysis of Pax8 expression during pancreogenesis and in adult islets. Immunohistochemical analysis using the most employed Pax8 polyclonal antibody revealed strong nuclear staining in the developing mouse pancreas and in mature human and mouse islets. Astonishingly, Pax8 mRNA in mouse islets was undetectable while human islets exhibited low levels. These discrepancies raised the possibility of antibody cross-reactivity. This premise was confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and immunohistochemistry. Thus, in islets polyclonal Pax8 staining corresponds mainly to Pax6. In order to circumvent this caveat, a novel Pax8 monoclonal antibody was used to re-evaluate whether Pax8 was indeed expressed in islets. Surprisingly, Pax8 was not detected in neither the developing pancreas or in mature islets. Reappraisal of pancreatic neuroendocrine tumors using this Pax8 monoclonal antibody exhibited no immunostaining as compared to the Pax8 polyclonal antibody. In conclusion, Pax8 is not expressed in the pancreas and cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker.

Thyroid Hormone-Regulated Expression of RC3/Neurogranin in the Immortalized Hypothalamic Cell Line GT1-7

Abstract: The calmodulin‐binding, protein kinase C substrate RC3/neurogranin is the product of a neuron‐specific gene expressed in the forebrain that is under specific regional and temporal control by thyroid hormone (3,5,3′‐triiodothyronine, T3). In vivo, some neuronal populations are sensitive and others are insensitive to T3. The goal of this study was to identify neuronal cell cultures that express RC3/neurogranin, to check whether they are sensitive to T3, and to examine the mechanism of regulation. We found that RC3 is induced by T3 in the hypothalamic cell line GT1‐7 at the transcriptional level. The half‐life of the mature mRNA was 20 h and was not affected by the hormone. Addition of T3 to the cell culture induces neurogranin mRNA after 6 h in the absence of new protein synthesis. These results suggest a direct transcriptional effect of T3 mediated through nuclear receptors. Indeed, GT1‐7 cells express functional T3 receptors, as shown by northern blotting, nuclear T3‐binding assays, and transactivation of reporter genes. The role of retinoic acid and glucocorticoids on RC3 expression was also evaluated, because we have previously noted the presence of consensus response elements for these hormones in the RC3 upstream promoter region. In contrast to T3, neither retinoic acid nor dexamethasone influences neurogranin expression despite the presence of respective functional receptors.

In Vivo Conditional Pax4 Overexpression in Mature Islet β-Cells Prevents Stress-Induced Hyperglycemia in Mice

OBJECTIVE To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in β-cells. RESEARCH DESIGN AND METHODS Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR. RESULTS Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. CONCLUSIONS Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

The liver receptor homolog-1 (LRH-1) is expressed in human islets and protects β-cells against stress-induced apoptosis

Liver receptor homolog (LRH-1) is an orphan nuclear receptor (NR5A2) that regulates cholesterol homeostasis and cell plasticity in endodermal-derived tissues. Estrogen increases LRH-1 expression conveying cell protection and proliferation. Independently, estrogen also protects isolated human islets against cytokine-induced apoptosis. Herein, we demonstrate that LRH-1 is expressed in islets, including β-cells, and that transcript levels are modulated by 17β-estradiol through the estrogen receptor (ER)α but not ERβ signaling pathway. Repression of LRH-1 by siRNA abrogated the protective effect conveyed by estrogen on rat islets against cytokines. Adenoviral-mediated overexpression of LRH-1 in human islets did not alter proliferation but conferred protection against cytokines and streptozotocin-induced apoptosis. Expression levels of the cell cycle genes cyclin D1 and cyclin E1 as well as the antiapoptotic gene bcl-xl were unaltered in LRH-1 expressing islets. In contrast, the steroidogenic enzymes CYP11A1 and CYP11B1 involved in glucocorticoid biosynthesis were both stimulated in transduced islets. In parallel, graded overexpression of LRH-1 dose-dependently impaired glucose-induced insulin secretion. Our results demonstrate the crucial role of the estrogen target gene nr5a2 in protecting human islets against-stressed-induced apoptosis. We postulate that this effect is mediated through increased glucocorticoid production that blunts the pro-inflammatory response of islets.

Thyroid Hormone-Dependent Regulation of Tα1 α-Tubulin during Brain Development

Thyroid hormone (T3) is essential for brain development and most of its actions are exerted at the gene expression level after interaction with nuclear receptors. In particular, genes encoding cytoskeletal proteins are influenced by the thyroidal status. Thyroid hormone is involved in the normal downregulation of the Talpha1 alpha-tubulin gene during postnatal growth. The action of T3 on Talpha1 tubulin expression is complex and is exerted at least at two levels. In cultured cells, T3 induces a transient and fast decrease of Talpha1 mRNA concentration. This effect is enhanced when transcription is blocked by actinomycin D, suggesting that T3 increases mRNA degradation. In transgenic animals T3 affects the expression of beta-galactosidase under control of the Talpha1 promoter in the same way as the endogenous gene, supporting an effect mediated through the Talpha1 promoter. However, the Talpha1 promoter is not regulated by T3 in transfected cells and, therefore, the effects of the hormone in vivo are likely to be indirect. It is concluded that regulation of Talpha1 alpha-tubulin by thyroid hormone is the result of multiple influences including effects on mRNA half life and indirect effects at the promoter level.

Pax8 detection in well-differentiated pancreatic endocrine tumors: how reliable is it?

To the Editor: One of the most challenging areas in pancreatic endocrine tumor (PET) biology is the identification of specific biomarkers that can morphologically and histochemically distinguish well-differentiated PETs from other types of neuroendocrine tumors (NETs). Novel markers could improve diagnosis accuracy and could consequently lead to a more appropriate and tailored treatment and therefore to a better prognosis. This is highly relevant from the clinical point of view as at least 10% of patients with NETs have a widespread disease of unknown primary origin, and available antineoplastic agents are fundamentally effective only on tumors of pancreatic origin. Unfortunately, current markers including synaptophysin and chromogranin A are unable to distinguish whether a particular NET originated initially from the endocrine pancreas or from other sites such as the gastrointestinal tract or the bronchopulmonary system. In this regard, a recent study published in the American Journal of Surgical Pathology has identified the transcription factor Pax8 as a potentially new biomarker capable of performing such a task. Although previously not detected in the pancreas, Long and colleagues reported strong Pax8 immunostaining in normal human islets and have suggested that this transcription factor could be a specific marker of mature pancreatic endocrine cells and could even be implicated in terminal differentiation of islet cells during development. This premise was corroborated by a second American Journal of Surgical Pathology publication confirming the expression of Pax8 in healthy human islets. The discovery of Pax8 expression in the endocrine pancreas led Long and colleagues to assess whether Pax8 was expressed in PETs and in other well-differentiated NETs and to evaluate its potential value as a specific biomarker. They showed that a significant percentage of primary welldifferentiated PETs displayed strong Pax8 staining, whereas ileal and pulmonary carcinoid tumors were negative. Furthermore, liver metastases of PET origin were often Pax8 positive. The investigators concluded that Pax8 could be a useful marker to determine the primary origin site of metastatic well-differentiated PETs lodged within the liver. Interestingly, an independent study substantiated these findings and proposed a flowchart in which Pax8 was included in a panel of immunohistochemical stainings to determine the site of origin of metastatic well-differentiated NET in the liver. Unfortunately, we would like to communicate to the American Journal of Surgical Pathology readership that the conclusion of these studies is potentially inaccurate and that Pax8 proteins are not detectable in either healthy islets or PETs. Indeed, subsequent to the publication by Long et al we embarked on a study to determine whether Pax8 could also be expressed during pancreatic islet development, thereby pinpointing to a yet unknown function of this transcription factor in islet physiology. To this end, we analyzed Pax8 expression during mouse pancreatic development and in adult islets by immunohistochemistry using the same Pax8 polyclonal antibody (ProteinTech Group Inc., Chicago, IL; Cat. No. 10336-1AP) that Long et al used in their study. It is noteworthy that this polyclonal antibody was also used for most, if not all, clinical studies that demonstrated Pax8 staining in PETs. Consistent with these published reports we were successful in detecting Pax8 immunostaining in mature mouse and human islets and in the developing mouse pancreas. However, in an attempt to complement our immunohistochemical data with transcript expression profile, we unexpectedly found that Pax8 mRNA levels in both human and mouse islets were low to undetectable. These findings led us to question the specificity of the Pax8 polyclonal antibody. Indeed, as the 9 Pax members share a high degree of homology at the N-terminal paired domain, the region included in the peptide use for originating the antibody, a potential cross-reactivity of the antibody to another Pax member could be envisaged. This premise was indeed confirmed by demonstrating that the polyclonal Pax8 antibody also recognized the islet-enriched Pax6 protein both by Western blotting and by immunohistochemistry. To circumvent this caveat, a novel Pax8 monoclonal antibody from Abcam (No. ab53490) generated against the more variable C-terminal region of Pax8 was used to reevaluate the expression pattern of Pax8 during development and in islets. This antibody recognizes the 2 predominant isoforms of Pax8 expressed in most tissues and tumors. Consistent with our QT-RTPCR data, neither the developing pancreas nor mature islets exhibited staining with this novel Pax8 monoclonal antibody as compared with the polyclonal antibody. These results led us to reassess whether Pax8 was truly present in PETs. To this end, serial sections of PETs were immunostained with Pax8 antibodies (polyclonal and monoclonal) and with anti-Pax6 sera (Fig. 1). Immunohistochemical analysis revealed positive staining both in nonneoplastic islets and in tumor areas of pancreas sections when the Pax8 polyclonal antibody was used, whereas no staining was revealed with the Pax8 monoclonal antibody (Figs. 1A, B). An immunostaining pattern similar to that of the Pax8 polyclonal antibody was discerned with the Pax6 antibody, substantiating cross-reactivity of the commonly used Pax8 polyclonal antibody with Pax6 and possibly with other members of the Pax family (Fig. 1C). Thus, in contrast to the study by Long et al, we LETTER TO THE EDITOR

PAX4 Defines an Expandable β-Cell Subpopulation in the Adult Pancreatic Islet

PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets.

The Diabetes-Linked Transcription Factor PAX4: From Gene to Functional Consequences

Paired box 4 (PAX4) is a key factor in the generation of insulin producing β-cells during embryonic development. In adult islets, PAX4 expression is sequestered to a subset of β-cells that are prone to proliferation and more resistant to stress-induced apoptosis. The importance of this transcription factor for adequate pancreatic islets functionality has been manifested by the association of mutations in PAX4 with the development of diabetes, independently of its etiology. Overexpression of this factor in adult islets stimulates β-cell proliferation and increases their resistance to apoptosis. Additionally, in an experimental model of autoimmune diabetes, a novel immunomodulatory function for this factor has been suggested. Altogether these data pinpoint at PAX4 as an important target for novel regenerative therapies for diabetes treatment, aiming at the preservation of the remaining β-cells in parallel to the stimulation of their proliferation to replenish the β-cell mass lost during the progression of the disease. However, the adequate development of such therapies requires the knowledge of the molecular mechanisms controlling the expression of PAX4 as well as the downstream effectors that could account for PAX4 action.

Targeting pancreatic expressed PAX genes for the treatment of diabetes mellitus and pancreatic neuroendocrine tumors.

ABSTRACT Introduction: Four members of the PAX family, PAX2, PAX4, PAX6 and PAX8 are known to be expressed in the pancreas. Accumulated evidences indicate that several pancreatic expressed PAX genes play a significant role in pancreatic development/functionality and alterations in these genes are involved in the pathogenesis of pancreatic diseases. Areas covered: In this review, we summarize the ongoing research related to pancreatic PAX genes in diabetes mellitus and pancreatic neuroendocrine tumors. We dissect the current knowledge at different levels; from mechanistic studies in cell lines performed to understand the molecular processes controlled by pancreatic PAX genes, to in vivo studies using rodent models that over-express or lack specific PAX genes. Finally, we describe human studies associating variants on pancreatic-expressed PAX genes with pancreatic diseases. Expert opinion: Based on the current literature, we propose that future interventions to treat pancreatic neuroendocrine tumors and diabetes mellitus could be developed via the modulation of PAX4 and/or PAX6 regulated pathways.

Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice.

Thyroid hormones induce several changes in whole body metabolism that are known to improve metabolic homeostasis. However, adverse side effects have prevented its use in the clinic. In view of the promising effects of thyroid hormones, we investigated the effects of levothyroxine supplementation on glucose homeostasis.