Transforming Growth Factor-ß1 Regulates Peroxisomal Genes/Proteins via Smad Signaling in Idiopathic Pulmonary Fibrosis Fibroblasts and Transgenic Mouse Models.

Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-ß1 (TGF-ß1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-ß1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. In vitro cell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month-old transgenic mice expressing a constitutively active TGF-ß type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-ß type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-ß1-Smad signaling: interaction of the Smad3 transcription factor with the PEX13 gene in chromatin immunoprecipitation-on-chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-ß type II receptor knockout mice. Taken together, data from this study suggest that TGF-ß1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF.

Understanding the link between neurotropic viruses, BBB permeability, and MS pathogenesis.

Neurotropic viruses can infiltrate the CNS by crossing the blood-brain barrier (BBB) through various mechanisms including paracellular, transcellular, and "Trojan horse" mechanisms during leukocyte diapedesis. These viruses belong to several families, including retroviruses; human immunodeficiency virus type 1 (HIV-1), flaviviruses; Japanese encephalitis (JEV); and herpesviruses; herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and mouse adenovirus 1 (MAV-1). For entering the brain, viral proteins act upon the tight junctions (TJs) between the brain microvascular endothelial cells (BMECs). For instance, HIV-1 proteins, such as glycoprotein 120, Nef, Vpr, and Tat, disrupt the BBB and generate a neurotoxic effect. Recombinant-Tat triggers amendments in the BBB by decreasing expression of the TJ proteins such as claudin-1, claudin-5, and zona occludens-1 (ZO-1). Thus, the breaching of BBB has been reported in myriad of neurological diseases including multiple sclerosis (MS). Neurotropic viruses also exhibit molecular mimicry with several myelin sheath proteins, i.e., antibodies against EBV nuclear antigen 1 (EBNA1) aa411-426 cross-react with MBP and EBNA1 aa385-420 was found to be associated with MS risk haplotype HLA-DRB1*150. Notably, myelin protein epitopes (PLP139-151, MOG35-55, and MBP87-99) are being used to generate model systems for MS such as experimental autoimmune encephalomyelitis (EAE) to understand the disease mechanism and therapeutics. Viruses like Theiler's murine encephalomyelitis virus (TMEV) are also commonly used to generate EAE. Altogether, this review provide insights into the viruses' association with BBB leakiness and MS along with possible mechanistic details which could potentially use for therapeutics.

2-(3-(Chloromethyl)benzoyloxy)benzoic Acid reduces prostaglandin E-2 concentration, NOX2 and NFKB expression, ROS production, and COX-2 expression in lipopolysaccharide-induced mice.

INTRODUCTION: Inflammation is a fundamental response to various insults, including microbial invasion and tissue injury. While aspirin (ASA) has been widely used for its anti-inflammatory properties, its adverse effects and limitations highlight the need for novel therapeutic alternatives. Recently, a novel salicylic acid derivative, 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid (3-CH2Cl), has emerged as a potential substitute for ASA, offering a simpler, environmentally friendly synthesis and a promising safety profile. AIM OF THE STUDY: This research aims to evaluate the anti-inflammatory mechanism of 3-CH2Cl in a lipopolysaccharide (LPS)-induced mouse model, focusing on its effects on prostaglandin E-2 (PGE-2) concentration, NOX2 and NFkB expression, ROS production, and COX-2 expression. MATERIAL AND METHODS: Utilizing BALB/C mice subjected to LPS-induced inflammation, we investigated the therapeutic potential of 3-CH2Cl. The study included synthesis and tablet preparation, experimental design, peripheral blood plasma PGE-2 measurement, splenocyte isolation and COX-2 expression analysis, nitric oxide and ROS measurement, and immunohistochemical analysis of NOX2 and NFkB expression. RESULTS: 3-CH2Cl significantly reduced PGE-2 levels (p = 0.005), NO concentration in liver homogenates (p = 0.005) and plasma (p = 0.0011), and expression of NOX2 and NFkB in liver (p < 0.0001) and splenocytes (p = 0.0036), demonstrating superior anti-inflammatory activity compared to ASA. Additionally, it showed potential in decreasing COX-2 expression in splenocytes. CONCLUSION: 3-CH2Cl exhibits potent anti-inflammatory properties, outperforming ASA in several key inflammatory markers in an LPS-induced inflammation model. The reduction of COX-2 expression, alongside the reduction of pro-inflammatory cytokines and oxidative stress markers, suggest it as a promising therapeutic agent for various inflammatory conditions.

Modulation of peroxisomal compartment by Epstein-Barr virus.

Viruses utilize clever strategies of interacting with various cellular factors, to remodel an organelle function, for the establishment of successful infection. In recent decades, numerous studies revealed the exploitation of the peroxisomal compartment by viruses. Epstein-Barr virus (EBV) is a ubiquitous virus linked with various cancers and neurological disorders. Till now, there is no report regarding the impacts of EBV infection on peroxisomal compartment. Therefore, we investigate the modulation of peroxisomal proteins in EBV transformed cell lines and during acute EBV infection. EBV positive Burkitt lymphoma cells of different origins as EBV transformed cells along with EBV negative Burkitt lymphoma cells as a control were used in this study. For acute EBV infection experiments, we infected peripheral blood mononuclear cells with EBV for three days. Thereafter, analyzed the gene expression patterns of peroxisomal proteins using qPCR. In addition, quantification of lipid content was performed by using fluorescence microscopy and biochemical assay. Our results revealed that, the peroxisomal proteins were distinctly regulated in EBV transformed cells and during acute EBV infection. Interestingly, PEX19 was significantly upregulated in EBV infected cells. Further, in correlation with the altered expression of peroxisomes proteins involved in lipid metabolism, the EBV transformed cells showed lower lipid abundance. Conversely, the lipid levels were increased during acute EBV infection. Our study highlights the importance of investigating the manipulation of the peroxisomal compartment by putting forward various differentially expressed proteins upon EBV infection. This study provides a base for further investigation to delve deeper into EBV and peroxisomal interactions. The future research in this direction could provide involvement of novel signaling pathways to understand molecular changes during EBV mediated pathologies.

Peroxisomes Are Highly Abundant and Heterogeneous in Human Parotid Glands.

The parotid gland is one of the major salivary glands producing a serous secretion, and it plays an essential role in the digestive and immune systems. Knowledge of peroxisomes in the human parotid gland is minimal; furthermore, the peroxisomal compartment and its enzyme composition in the different cell types of the human parotid gland have never been subjected to a detailed investigation. Therefore, we performed a comprehensive analysis of peroxisomes in the human parotid gland's striated duct and acinar cells. We combined biochemical techniques with various light and electron microscopy techniques to determine the localization of parotid secretory proteins and different peroxisomal marker proteins in parotid gland tissue. Moreover, we analyzed the mRNA of numerous gene encoding proteins localized in peroxisomes using real-time quantitative PCR. The results confirm the presence of peroxisomes in all striated duct and acinar cells of the human parotid gland. Immunofluorescence analyses for various peroxisomal proteins showed a higher abundance and more intense staining in striated duct cells compared to acinar cells. Moreover, human parotid glands comprise high quantities of catalase and other antioxidative enzymes in discrete subcellular regions, suggesting their role in protection against oxidative stress. This study provides the first thorough description of parotid peroxisomes in different parotid cell types of healthy human tissue.

Interferon Beta-1a versus Combined Interferon Beta-1a and Oligodendrocyte-Specific FGFR1 Deletion in Experimental Autoimmune Encephalomyelitis.

Recombinant beta interferons-1 (IFNß-1) are used as first line therapies in patients with relapsing multiple sclerosis (MS), a chronic inflammatory and neurodegenerative disease of the CNS. IFNß-1a/b has moderate effects on the prevention of relapses and slowing of disease progression. Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) are known to play a key role in the pathology of MS and its model EAE. To investigate the effects of short-term treatment with s.c. IFNß-1a versus the combined application of s.c. IFNß-1a and oligodendrocyte-specific deletion of FGFR1 (Fgfr1ind-/- mice) in MOG35-55-induced EAE. IFNß-1a (30 mg/kg) was applied s.c. from days 0-7 p.i. of EAE in controls and Fgfr1ind-/- mice. FGFR signaling proteins associated with inflammation/degeneration in MS/EAE were analyzed by western blot in the spinal cord. Further, FGFR1 in Oli-neu oligodendrocytes were inhibited by PD166866 and treated with IFNß-1a (400 ng/mL). Application of IFNß-1a over 8 days resulted in less symptoms only at the peak of disease (days 9-11) compared to controls. Application of IFNß-1a in Fgfr1ind-/- mice resulted in less symptoms primarily in the chronic phase of EAE. Fgfr1ind-/- mice treated with IFNß-1a showed increased expression of pERK and BDNF. In Oli-neu oligodendrocytes, treatment with PD166866 and IFNß-1a also showed an increased expression of pERK and BDNF/TrkB. These data suggest that the beneficial effects in the chronic phase of EAE and on signaling molecules associated with ERK and BDNF expression are caused by the modulation of FGFR1 and not by interferon beta-1a. FGFR may be a potential target for therapy in MS.

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Anti-inflammatory activity of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid in LPS-induced rat model.

INTRODUCTION: Salicylic acid derivate is very popular for its activity to suppress pain, fever, and inflammation. One of its derivatives is acetylsalicylic acid (ASA) which has been reported repeatedly that, as a non-steroidal anti-inflammatory drug (NSAID), it has a cardioprotective effect. Although ASA has various advantages, several studies have reported that it may induce severe peptic ulcer disease. We recently synthesized a new compound derived from salicylic acid, namely 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid (3-CH2Cl) which still has the benefit of acetylsalicylic acid as an analgesic and antiplatelet, but lacks its harmful side effects (Caroline et al., 2019). In addition, in silico studies of 3-CH2Cl showed a higher affinity towards protein receptor cyclooxygenase-2 (COX-2; PDB: 5F1A) than ASA. We hypothesized that 3-CH2Cl inhibits the COX-2 activity which could presumably decrease the inflammatory responses. However, no knowledge is available on the anti-inflammatory response and molecular signaling of this new compound. Hence, in this study, we investigated the potential functional relevance of 3-CH2Cl in regulating the inflammatory response in lipopolysaccharide (LPS)-induced rats. The results of this study show that this compound could significantly reduce the inflammatory parameter in LPS-induced rats. MATERIAL AND METHODS: Rats were induced with LPS of 0.5 mg/kg bw intravenously, prior oral administration with vehicle (3% Pulvis Gummi Arabicum / PGA), 500 mg/60 kg body weight (bw; rat dosage converted to human) of 3-CH2Cl and ASA. The inflammatory parameters such as changes in the temperature of septic shock, cardiac blood plasma concentrations of IL-1ß and TNF-α (ELISA), blood inflammation parameters, white blood cell concentrations, and lung histopathology were observed. Meanwhile, the stability of 3-CH2Cl powder was evaluated. RESULT: After the administration of 500 mg/60 kg bw of 3-CH2Cl (rat dosage converted to human) to LPS-induced rats, we observed a significant reduction of both TNF-α (5.70+/-1.04 × 103 pg/mL, p=<0.001) and IL-1ß (2.32+/-0.28 × 103 pg/mL, p=<0.001) cardiac blood plasma concentrations. Besides, we found a reduction of white blood cell concentration and the severity of lung injury in the 3-CH2Cl group compared to the LPS-induced rat group. Additionally, this compound maintained the rat body temperature within normal limits during inflammation, preventing the rats to undergo septic shock, characterized by hypothermic (t = 120 min.) or hyperthermic (t = 360 min) conditions. Furthermore, 3-CH2Cl was found to be stable until 3 years at 25°C with a relative humidity of 75 ± 5%. CONCLUSION: 3-CH2Cl compound inhibited inflammation in the LPS-induced inflammation response model in rats, hypothetically through binding to COX-2, and presumably inhibited LPS-induced NF-κß signaling pathways. This study could be used as a preliminary hint to investigate the target molecular pathways of 3-CH2Cl as a novel and less toxic therapeutical agent in alleviating the COX-related inflammatory diseases, and most importantly to support the planning and development of clinical trial.

2021 Update on Diagnostic Markers and Translocation in Salivary Gland Tumors.

Salivary gland tumors are a rare tumor entity within malignant tumors of all tissues. The most common are malignant mucoepidermoid carcinoma, adenoid cystic carcinoma, and acinic cell carcinoma. Pleomorphic adenoma is the most recurrent form of benign salivary gland tumor. Due to their low incidence rates and complex histological patterns, they are difficult to diagnose accurately. Malignant tumors of the salivary glands are challenging in terms of differentiation because of their variability in histochemistry and translocations. Therefore, the primary goal of the study was to review the current literature to identify the recent developments in histochemical diagnostics and translocations for differentiating salivary gland tumors.

Differential Expression of Peroxisomal Proteins in Distinct Types of Parotid Gland Tumors.

Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.

Oligodendrocyte-Specific Deletion of FGFR1 Reduces Cerebellar Inflammation and Neurodegeneration in MOG35-55-Induced EAE.

Multiple sclerosis (MS) is a chronic inflammatory and degenerative disease of the central nervous system (CNS). MS commonly affects the cerebellum causing acute and chronic symptoms. Cerebellar signs significantly contribute to clinical disability, and symptoms such as tremor, ataxia, and dysarthria are difficult to treat. Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in demyelinating pathologies such as MS. In autopsy tissue from patients with MS, increased expression of FGF1, FGF2, FGF9, and FGFR1 was found in lesion areas. Recent research using mouse models has focused on regions such as the spinal cord, and data on the expression of FGF/FGFR in the cerebellum are not available. In recent EAE studies, we detected that oligodendrocyte-specific deletion of FGFRs results in a milder disease course, less cellular infiltrates, and reduced neurodegeneration in the spinal cord. The objective of this study was to characterize the role of FGFR1 in oligodendrocytes in the cerebellum. Conditional deletion of FGFR1 in oligodendrocytes (Fgfr1ind-/-) was achieved by tamoxifen application, EAE was induced using the MOG35-55 peptide. The cerebellum was analyzed by histology, immunohistochemistry, and western blot. At day 62 p.i., Fgfr1ind-/- mice showed less myelin and axonal degeneration compared to FGFR1-competent mice. Infiltration of CD3(+) T cells, Mac3(+) cells, B220(+) B cells and IgG(+) plasma cells in cerebellar white matter lesions (WML) was less in Fgfr1ind-/-mice. There were no effects on the number of OPC or mature oligodendrocytes in white matter lesion (WML). Expression of FGF2 and FGF9 associated with less myelin and axonal degeneration, and of the pro-inflammatory cytokines IL-1ß, IL-6, and CD200 was downregulated in Fgfr1ind-/- mice. The FGF/FGFR signaling protein pAkt, BDNF, and TrkB were increased in Fgfr1ind-/- mice. These data suggest that cell-specific deletion of FGFR1 in oligodendrocytes has anti-inflammatory and neuroprotective effects in the cerebellum in the EAE disease model of MS.

Influence of gender in monocrotaline and chronic hypoxia induced pulmonary hypertension in obese rats and mice.

BACKGROUND: Obesity and pulmonary hypertension (PH) share common characteristics, such as augmented inflammation and oxidative stress. However, the exact role of obesity in the pathology of PH is largely uninvestigated. Therefore, we have hypothesized that in the context of obesity the gender difference may have influence on development of PH in animal models of this disease. METHODS: Animal experiments were conducted in monocrotaline (MCT) and chronic hypoxia (HOX) models of PH. Lean and obese Zucker rats or B6 mice of both genders were used for MCT or HOX models, respectively. Echocardiography, hemodynamic measurements, histology and immuno-histochemistry were performed to analyze various parameters, such as right ventricular function and hypertrophy, hemodynamics, pulmonary vascular remodeling and lung inflammation. RESULTS: Both lean and obese male and female Zucker rats developed PH after a single MCT injection. However, negligible differences were seen between lean and obese male rats in terms of PH severity at the end stage of disease. Conversely, a more prominent and severe PH was observed in obese female rats compared to their lean counterparts. In contrast, HOX induced PH in lean and obese, male and female mice did not show any apparent differences. CONCLUSION: Gender influences PH severity in obese MCT-injected rats. It is also an important factor associated with altered inflammation. However, further research is necessary to investigate and reveal the underlying mechanisms.

A New Immunomodulatory Role for Peroxisomes in Macrophages Activated by the TLR4 Ligand Lipopolysaccharide.

Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene Pex14 or Mfp2 markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation.

Compromised peroxisomes in idiopathic pulmonary fibrosis, a vicious cycle inducing a higher fibrotic response via TGF-ß signaling.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-ß) receptor II knockout mice indicating a role for TGF-ß signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-ß1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-ß signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-ß) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-ß-induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.

PEX6 is Expressed in Photoreceptor Cilia and Mutated in Deafblindness with Enamel Dysplasia and Microcephaly.

Deafblindness is part of several genetic disorders. We investigated a consanguineous Egyptian family with two siblings affected by congenital hearing loss and retinal degeneration, initially diagnosed as Usher syndrome type 1. At teenage, severe enamel dysplasia, developmental delay, and microcephaly became apparent. Genome-wide homozygosity mapping and whole-exome sequencing detected a homozygous missense mutation, c.1238G>T (p.Gly413Val), affecting a highly conserved residue of peroxisomal biogenesis factor 6, PEX6. Biochemical profiling of the siblings revealed abnormal and borderline plasma phytanic acid concentration, and cerebral imaging revealed white matter disease in both. We show that Pex6 localizes to the apical extensions of secretory ameloblasts and differentiated odontoblasts at early stages of dentin synthesis in mice, and to cilia of retinal photoreceptor cells. We propose PEX6, and possibly other peroxisomal genes, as candidate for the rare cooccurrence of deafblindness and enamel dysplasia. Our study for the first time links peroxisome biogenesis disorders to retinal ciliopathies.

C22-bronchial and T7-alveolar epithelial cell lines of the immortomouse are excellent murine cell culture model systems to study pulmonary peroxisome biology and metabolism.

In pulmonary research, temperature-sensitive immortalized cell lines derived from the lung of the "immortomouse" (H-2k(b)-tsA58 transgenic mouse), such as C22 club cells and T7 alveolar epithelial cells type II (AECII), are frequently used cell culture models to study CC10 metabolism and surfactant synthesis. Even though peroxisomes are highly abundant in club cells and AECII and might fulfill important metabolic functions therein, these organelles have never been investigated in C22 and T7 cells. Therefore, we have characterized the peroxisomal compartment and its associated gene transcription in these cell lines. Our results show that peroxisomes are highly abundant in C22 and T7 cells, harboring a common set of enzymes, however, exhibiting specific differences in protein composition and gene expression patterns, similar to the ones observed in club cells and AECII in situ in the lung. C22 cells contain a lower number of larger peroxisomes, whereas T7 cells possess more numerous tubular peroxisomes, reflected also by higher levels of PEX11 proteins. Moreover, C22 cells harbor relatively higher amounts of catalase and antioxidative enzymes in distinct subcellular compartments, whereas T7 cells exhibit higher levels of ABCD3 and plasmalogen synthesizing enzymes as well as nuclear receptors of the PPAR family. This study suggest that the C22 and T7 cell lines of the immortomouse lung are useful models to study the regulation and metabolic function of the peroxisomal compartment and its alterations by paracrine factors in club cells and AECII.

Postnatal development of the bronchiolar club cells of distal airways in the mouse lung: stereological and molecular biological studies.

Club (Clara) cells are nonciliated secretory epithelial cells present in bronchioles of distal pulmonary airways. So far, no information is available on the postnatal differentiation of club cells by a combination of molecular biological, biochemical, and stereological approaches in the murine lung. Therefore, the present study was designed to investigate the changes in the club cell secretory proteins (CC10, surfactant proteins A, B and D) and club cell abundance within the epithelium of bronchioles of distal airways during the postnatal development of the mouse lung. Perfusion-fixed murine lungs of three developmental stages (newborn, 15-day-old and adult) were used. Frozen, unfixed lungs were used for cryosectioning and subsequent laser-assisted microdissection of bronchiolar epithelial cells and RT-PCR analyses. High resolution analyses of the three-dimensional structures and composition of lung airways were obtained by scanning electron microscopy. Finally, using design-based stereology, the total and average club cell volume and the volume of secretory granules were quantified by light and transmission electron microscopy. Our results reveal that murine club cells are immature at birth and differentiate postnatally. Further, increase of the club cell volume and number of intracellular granules are closely correlated to the total lung volume enlargement. However, secretory granule density was only increased within the first 15 days of postnatal development. The differentiation is accompanied by a decrease in glycogen content, and a close positive relationship between CC10 expression and secretory granule abundance. Taken together, our data are consistent with the concept that the morphological and functional differentiation of club cells is a postnatal phenomenon.

Mammalian SOD2 is exclusively located in mitochondria and not present in peroxisomes.

Superoxide dismutases (SODs) are metalloenzymes that belong to the essential antioxidant enzyme systems of virtually all oxygen-respiring organisms. SODs catalyze the dismutation of highly reactive superoxide radicals into hydrogen peroxide and molecular oxygen. For the subcellular localization of the manganese superoxide dismutase (SOD2) in eukaryotic cells, a dual mitochondrial localization and peroxisomal localization were proposed in the literature. However, our own observation from immunofluorescence preparations of human and mouse tissues suggested that SOD2 serves as an excellent marker protein for mitochondria but never co-localized with peroxisomes. To clarify whether our observations were correct, we have carefully reinvestigated the subcellular localization of SOD2 using sensitive double-immunofluorescence methods on frozen and paraffin sections as well as in cell culture preparations. In addition, ultrastructural analyses were performed with post-embedding immunoelectron microscopy on LR White sections as well as labeling of ultrathin cryosections with various immunogold techniques. In all morphological experiments, the SOD2 localization was compared to one of the catalase, a typical marker protein for peroxisomes, solely localized in these organelles. Moreover, biochemical subcellular fractions of mouse liver was used to isolate enriched organelles and highly purified peroxisomal fractions for Western blot analyses of the exact subcellular distributions of SOD2 and catalase. All results with the various methodologies, tissues, and cell types used revealed that catalase and SOD2 were always confined to distinct and separate subcellular compartments. SOD2 was unequivocally in mitochondria, but never present in peroxisomes. Furthermore, our results are supported by accumulating database information on organelle proteomes that also indicate that SOD2 is a pure mitochondrial protein.

Peroxisomes in dental tissues of the mouse.

Patients with mild forms of peroxisomal biogenesis disorders show facial dysmorphism and exhibit dentition problems accompanied by enamel hypoplasia. However, no information is available on the role of peroxisomes in dental and paradontal tissues. Therefore, we studied the distribution of these organelles, their protein composition and the expression of corresponding genes during dental development and in mature decalcified teeth in mice. Perfusion-fixed heads of mice of different developmental stages (E13.5 to adult) were cut in sagittal direction into two halves and embedded in paraffin for serial sectioning and subsequent peroxidase-based immunohistochemistry or double-immunofluorescence preparations. Frozen, unfixed heads of newborn mice were used for cryosectioning and subsequent laser-assisted microdissection of ameloblasts and odontoblasts, RNA isolation and RT-PCR analysis. Our results revealed the presence of peroxisomes already in the bud stage of dental development. An increase in peroxisome abundance was noted during differentiation of ameloblasts and odontoblasts with the highest number of organelles in Tomes' processes of mature ameloblasts. A strong heterogeneity of peroxisomal enzyme content developed within differentiated dental cell types. A drastic down-regulation of catalase in maturing ameloblasts was noted in contrast to high levels of lipid metabolizing enzymes in peroxisomes of these cells. As known from the literature, differentiated ameloblasts are more prone to oxidative damage which could be explained by the low catalase levels inside of this cell type.

The biogenesis protein PEX14 is an optimal marker for the identification and localization of peroxisomes in different cell types, tissues, and species in morphological studies.

Catalase and ABCD3 are frequently used as markers for the localization of peroxisomes in morphological experiments. Their abundance, however, is highly dependent on metabolic demands, reducing the validity of analyses of peroxisomal abundance and distribution based solely on these proteins. We therefore attempted to find a protein which can be used as an optimal marker for peroxisomes in a variety of species, tissues, cell types and also experimental designs, independently of peroxisomal metabolism. We found that the biogenesis protein peroxin 14 (PEX14) is present in comparable amounts in the membranes of every peroxisome and is optimally suited for immunoblotting, immunohistochemistry, immunofluorescence, and immunoelectron microscopy. Using antibodies against PEX14, we could visualize peroxisomes with almost undetectable catalase content in various mammalian tissue sections (submandibular and adrenal gland, kidney, testis, ovary, brain, and pancreas from mouse, cat, baboon, and human) and cell cultures (primary cells and cell lines). Peroxisome labeling with catalase often showed a similar tissue distribution to the mitochondrial enzyme mitochondrial superoxide dismutase (both responsible for the degradation of reactive oxygen species), whereas ABCD3 exhibited a distinct labeling only in cells involved in lipid metabolism. We increased the sensitivity of our methods by using QuantumDots™, which have higher emission yields compared to classic fluorochromes and are unsusceptible to photobleaching, thereby allowing more exact quantification without artificial mistakes due to heterogeneity of individual peroxisomes. We conclude that PEX14 is indeed the best marker for labeling of peroxisomes in a variety of tissues and cell types in a consistent fashion for comparative morphometry.