Thyroidal Transcriptomic Profiles of Pathoadaptive Responses to Congenital Hypothyroidism in XB130 Knockout Mice.

Congenital hypothyroidism is a genetic condition in which the thyroid gland fails to produce sufficient thyroid hormone (TH), resulting in metabolic dysfunction and growth retardation. Xb130-/- mice exhibit perturbations of thyrocyte cytoskeleton and polarity, and develop postnatal transient growth retardation due to congenital hypothyroidism, leading ultimately to multinodular goiter. To determine the underlying mechanisms, we performed transcriptomic analyses on thyroid glands of mice at three age points: week 2 (W2, before visible growth retardation), W4 (at the nadir of growth); and W12 (immediately before full growth recovery). Using gene set enrichment analysis, we compared a defined set of thyroidal genes between Xb130+/+ and Xb130-/- mice to identify differentially enriched gene clusters. At the earliest postnatal stage (W2), the thyroid glands of Xb130-/- mice exhibited significantly downregulated gene clusters related to cellular metabolism, which continued to W4. Additionally, mutant thyroids at W4 and W12 showed upregulated gene clusters related to extracellular matrix, angiogenesis, and cell proliferation. At W12, despite nearly normal levels of serum TH and TSH and body size, a significantly large number of gene clusters related to inflammatory response were upregulated. Early postnatal TH deficiency may suppress cellular metabolism within the thyroid gland itself. Upregulation of genes related to extracellular matrix and angiogenesis may promote subsequent thyroid growth. Chronic inflammatory responses may contribute to the pathogenesis of multinodular goiter in later life. Some of the pathoadaptive responses of Xb130-/- mice may overlap with those from other mutations causing congenital hypothyroidism.

Pathogenesis of Multinodular Goiter in Elderly XB130-Deficient Mice: Alteration of Thyroperoxidase Affinity with Iodide and Hydrogen Peroxide.

Background: Multinodular goiter (MNG) is the most common disorder of the thyroid gland. Aging and genetic mutations that impair thyroid hormone (TH) production have been implicated in the development of MNG. XB130 is an adaptor/scaffold protein predominantly expressed in the thyroid gland. XB130 deficiency leads to transient postnatal growth retardation in mice due to congenital hypothyroidism. We studied the formation of MNG and possible mechanisms in elderly mice. Methods: Thyroid glands of male and female Xb130-knockout (Xb130-/-), heterozygous (Xb130+/-), and wild-type (Xb130+/+) mice at the ages of 12-20 months were harvested for visual examination, histopathological, and immunohistological analyses. Blood and thyroid samples were collected after feeding elderly mice with a low iodine diet for 125I uptake and perchlorate discharge assay. The activity of thyroperoxidase (Tpo) was examined by spectrophotometric evaluation of iodide oxidation. Results: While moderate MNG was seen in Xb130+/+ and Xb130+/- mice, severe MNG, characterized by multiple nodules intermixed with dilated colloid-rich macrofollicles, was found only in Xb130-/- mice at 18 months. Thyrocyte cytoskeletal structure and cell adhesion molecules were disorganized, and TH production was significantly reduced. Reduced iodide organification was seen in elderly Xb130+/+ mice and further enhanced in Xb130-/- mice. In Xb130+/+ mice, Tpo shows high affinity with hydrogen peroxide (H2O2) throughout aging, but reduced affinity with iodide in an age-dependent manner. By contrast, in elderly Xb130-/- mice, the affinity of Tpo for iodide remained high, but the affinity of Tpo for H2O2 was reduced. Conclusions: The pathophysiological features in the thyroid glands of aged Xb130-/- mice closely resemble the features of MNG in humans. Moderate MNG in elderly mice was dramatically aggravated by XB130 deficiency. Reduced affinity of Tpo for H2O2 may contribute to MNG development via oxidative stress. This could be specific to XB130 deficiency but also could be a common mechanism in MNG. Its clinical relevance should be further investigated.

XB130 Deficiency Causes Congenital Hypothyroidism in Mice due to Disorganized Apical Membrane Structure and Function of Thyrocytes.

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130-/- mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130-/- mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130-/- mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130-/- mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism.

XB130 deficiency enhances carcinogen-induced skin tumorigenesis.

XB130 is an adaptor protein that functions as a mediator of multiple tyrosine kinases important for regulating cell proliferation, survival, migration and invasion. Formerly predicted as an oncogene, alterations of its expression are documented in various human cancers. However, the exact role of XB130 in tumorigenesis is unknown. To address its function in skin tumorigenesis, a two-stage dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA) study was performed on XB130 knockout (KO), heterozygous (HZ) and wild-type (WT) littermate mice. DMBA/TPA-treated XB130 KO and HZ males developed a significantly higher number of epidermal tumors that were notably larger in size than did WT mice. Interestingly, DMBA/TPA-treated female mice did not show any difference in tumor multiplicity regardless of the genotypes. The skin tumor lesions of XB130 KO males were more progressed with an increased frequency of keratoacanthoma. Deficiency of XB130 dramatically increased epidermal tumor cell proliferation. The responses to DMBA and TPA stimuli were also individually investigated to elucidate the mechanistic role of XB130 at different stages of tumorigenesis. DMBA-treated male XB130 KO mice showed compensatory p53-mediated stress response. TPA-treated XB130 KO males demonstrated more skin ulceration with more severe edema, enhanced cell proliferation, accumulation of infiltrating neutrophils and increased production of pro-inflammatory cytokine genes compared with WT mice. Enhanced activities of nuclear factor-kappa B pathway, increased protein expression of metalloproteinase-9 and ERK1/2 phosphorylation were found in these KO mice. These findings demonstrate that XB130 acts as a tumor suppressor in carcinogen-induced skin tumorigenesis that may be mediated through inhibiting inflammation.

Pentraxin 3 deficiency enhances features of chronic rejection in a mouse orthotopic lung transplantation model.

Chronic lung allograft dysfunction (CLAD) is a serious complication after lung transplantation and thought to represent chronic rejection. Increased expression of Pentraxin 3 (PTX3), an acute phase protein, was associated with worse outcome in lung transplant patients. To determine the role of recipient PTX3 in development of chronic rejection, we used a minor alloantigen-mismatched murine orthotopic single lung transplant model. Male C57BL/10 mice were used as donors. Male PTX3 knockout (KO) mice and their wild type (WT) littermates on 129/SvEv/C57BL6/J background were used as recipients. In KO recipients, 7/13 grafted lungs were consolidated without volume recovery on CT scan, while only 2/9 WT mice showed similar graft consolidation. For grafts where lung volume could be reliably analyzed by CT scan, the lung volume recovery was significantly reduced in KO mice compared to WT. Interstitial inflammation, parenchymal fibrosis and bronchiolitis obliterans scores were significantly higher in KO mice. Presence of myofibroblasts and lymphoid aggregation was significantly enhanced in the grafts of PTX3 KO recipients. Recipient PTX3 deficiency enhanced chronic rejection-like lesions by promoting a fibrotic process in the airways and lung parenchyma. The underlying mechanisms and potential protective role of exogenous PTX3 as a therapy should be further explored.

XB130 deficiency enhances lipopolysaccharide-induced septic response and acute lung injury.

XB130 is a novel oncoprotein that promotes cancer cell survival, proliferation and migration. Its physiological function in vivo is largely unknown. The objective of this study was to determine the role of XB130 in lipopolysaccharide (LPS)-induced septic responses and acute lung injury. LPS was intraperitoneally administrated to Xb130 knockout (KO) and wild type (WT) mice. There was a significant weight loss in KO mice at Day 2 and significantly higher disease scores during the 7 days of observation. The levels of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, interleukin-6 and interleukin-10 in the serum were significantly higher in KO mice at Day 2. In KO mice there were a significantly higher lung injury score, higher wet/dry lung weight ratio, more apoptotic cells and less proliferative cells in the lung. Macrophage infiltration was significantly elevated in the lung of KO mice. There was significantly increased number of p-GSK-3ß positive cells in KO mice, which were mainly neutrophils and macrophages. XB130 is expressed in alveolar type I and type II cells in the lung. The expression in these cells was significantly reduced after LPS challenge. XB130 deficiency delayed the recovery from systemic septic responses, and the presence of XB130 in the alveolar epithelial cells may provide protective mechanisms by reducing cell death and promoting cell proliferation, and reducing pulmonary permeability.

XB130 promotes bronchioalveolar stem cell and Club cell proliferation in airway epithelial repair and regeneration.

Proliferation of bronchioalveolar stem cells (BASCs) is essential for epithelial repair. XB130 is a novel adaptor protein involved in the regulation of epithelial cell survival, proliferation and migration through the PI3K/Akt pathway. To determine the role of XB130 in airway epithelial injury repair and regeneration, a naphthalene-induced airway epithelial injury model was used with XB130 knockout (KO) mice and their wild type (WT) littermates. In XB130 KO mice, at days 7 and 14, small airway epithelium repair was significantly delayed with fewer number of Club cells (previously called Clara cells). CCSP (Club cell secreted protein) mRNA expression was also significantly lower in KO mice at day 7. At day 5, there were significantly fewer proliferative epithelial cells in the KO group, and the number of BASCs significantly increased in WT mice but not in KO mice. At day 7, phosphorylation of Akt, GSK-3ß, and the p85α subunit of PI3K was observed in airway epithelial cells in WT mice, but to a much lesser extent in KO mice. Microarray data also suggest that PI3K/Akt-related signals were regulated differently in KO and WT mice. An inhibitory mechanism for cell proliferation and cell cycle progression was suggested in KO mice. XB130 is involved in bronchioalveolar stem cell and Club cell proliferation, likely through the PI3K/Akt/GSK-3ß pathway.

Chromosome Condensation 1-Like (Chc1L) Is a Novel Tumor Suppressor Involved in Development of Histiocyte-Rich Neoplasms.

Human chromosomal region 13q14 is a deletion hotspot in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. This region is believed to host multiple tumor suppressors. Chromosome Condensation 1-like (CHC1L) is located at 13q14, and found within the smallest common region of loss of heterozygosity in prostate cancer. Decreased expression of CHC1L is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. However, there is no direct evidence for CHC1L's putative tumor suppressing role in current literature. Presently, we describe the generation and characterization of Chc1L knockout mice. Chc1L-/- mice do not develop cancer at a young age, but bone marrow and spleen cells from 8-12 week-old mice display an exaggerated proliferative response. By approximately two years of age, knockout and heterozygote mice have a markedly increased incidence of tumorigenesis compared to wild-type controls, with tumors occurring mainly in the spleen, mesenteric lymph nodes, liver and intestinal tract. Histopathological analysis found that most heterozygote and knockout mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. Our study suggests that Chc1L is involved in suppression of these two histiocyte-rich neoplasms in mice and supports clinical data suggesting that CHC1L loss of function is an important step in the pathogenesis of cancers containing 13q14 deletion.

XB130-A Novel Adaptor Protein: Gene, Function, and Roles in Tumorigenesis.

Several adaptor proteins have previously been shown to play an important role in the promotion of tumourigenesis. XB130 (AFAP1L2) is an adaptor protein involved in many cellular functions, such as cell survival, cell proliferation, migration, and gene and miRNA expression. XB130's functional domains and motifs enable its interaction with a multitude of proteins involved in several different signaling pathways. As a tyrosine kinase substrate, tyrosine phosphorylated XB130 associates with the p85 α regulatory subunit of phosphoinositol-3-kinase (PI3K) and subsequently affects Akt activity and its downstream signalling. Tumourigenesis studies show that downregulation of XB130 expression by RNAi inhibits tumor growth in mouse xenograft models. Furthermore, XB130 affects tumor oncogenicity by regulating the expression of specific tumour suppressing miRNAs. The expression level and pattern of XB130 has been studied in various human tumors, such as thyroid, esophageal, and gastric cancers, as well as, soft tissue tumors. Studies show the significant effects of XB130 in tumourigenesis and suggest its potential as a diagnostic biomarker and therapeutic target for cancer treatments.

α1-Antitrypsin inhibits ischemia reperfusion-induced lung injury by reducing inflammatory response and cell death.

BACKGROUND: Pulmonary ischemia-reperfusion (IR)-induced lung injury is a severe complication that increases the likelihood of primary graft dysfunction and early death after lung transplantation. Inflammatory cytokine release and cell death play a critical role in the development of IR-induced lung injury. α1-Antitrypsin (A1AT) is a protease inhibitor clinically used for the treatment of A1AT-deficiency emphysema. On the basis of a literature review, we hypothesize that A1AT may have the potential to reduce IR-induced lung injury through its anti-inflammatory and anti-apoptotic effects. METHODS: A human pulmonary cell culture model was used to simulate IR processes in lung transplantation. Effects of A1AT on cell death and cytokine production were examined. A rat pulmonary IR model, in which the left pulmonary hilum was clamped for 90 minutes, followed by reperfusion for 2 hours, was used to determine the effects of A1AT on acute lung injury, function, cell death, and inflammatory response. RESULTS: A1AT significantly inhibited cell death and inflammatory cytokine release dose-dependently in vitro and significantly improved lung oxygenation and lung mechanics and reduced pulmonary edema in vivo. Moreover, A1AT inhibited neutrophil infiltration in the lung and reduced cell death and significantly reduced IR-induced inflammatory mediators in plasma, including interleukin (IL)-1α, IL-4, IL-12p70, monocyte chemotactic protein 1, and tumor necrosis factor-α. CONCLUSIONS: Considering its current clinical use, our findings indicate that administration of A1AT may be an effective and safe therapy for the treatment of IR injury in human lung transplantation.