Transcription factor 21 (TCF21) promotes proinflammatory interleukin 6 expression and extracellular matrix remodeling in visceral adipose stem cells.

The visceral (VIS) and subcutaneous (SQ) fat pads are developmentally distinct white adipose tissue depots and contribute differently to inflammation and insulin resistance associated with obesity. The basic helix-loop-helix transcriptional regulator, transcription factor 21 (TCF21), is a marker gene for white adipose tissues and is abundantly expressed in VIS-derived adipose stem cells (ASCs), but not in SQ-derived ASCs. However, TCF21's role in regulating fat depot-specific gene expression and function is incompletely understood. Here, using siRNA-mediated Tcf21 knockdowns and lentiviral gene transfer of TCF21 in mouse ASCs, we demonstrate that TCF21 is required for the VIS ASC-specific expression of interleukin 6 (IL6), a key cytokine that contributes to the proinflammatory nature of VIS depots. Concurrently, TCF21 promotes MMP-dependent collagen degradation and type IV collagen deposition through the regulation of the extracellular matrix (ECM) modifiers, matrix metalloproteinase (MMP) 2, MMP13, and tissue inhibitor of MMP1 (TIMP1), as well as collagen type IV α1 chain (COL4A1) in VIS ASCs. We also found that although IL6 mediates the expression of Mmp13 and Timp1 in VIS ASCs, the TCF21-dependent expression of Mmp2 and Col4a1 is IL6-independent. These results suggest that TCF21 contributes to the proinflammatory environment in VIS fat depots and to active ECM remodeling of these depots by regulating IL6 expression and MMP-dependent ECM remodeling in a spatiotemporally coordinated manner.

Thyroglobulin (Tg) activates MAPK pathway to induce thyroid cell growth in the absence of TSH, insulin and serum.

The growth of thyroid cells is tightly regulated by thyroid stimulating hormone (TSH) through the cyclic adenosine 3', 5'-monophosphate (cAMP) signaling pathway by potentiating the mitogenic activity of insulin and insulin-like growth factors (IGFs). However, we recently reported that thyroglobulin (Tg), a major product of the thyroid, also induces the growth of thyroid cells cultured in 0.2% serum in the absence of TSH and insulin. In this report, we demonstrate that Tg induced phosphorylation of molecules of the c-Raf/MEK/ERK pathway of the mitogen-activated protein kinase (MAPK). The MEK-1/2 inhibitor PD98059 suppressed Tg-induced phosphorylation of ERK1/2 and reduced bromodeoxyuridine (BrdU) incorporation. Tg also induced expression of the essential transcriptional factors c-Myc, c-Fos and c-Jun and phosphorylation of the retinoblastoma (Rb) protein. The present results, together with the previous report, suggest that Tg utilizes multiple signaling cascades to induce thyroid cell growth independent of TSH/cAMP stimulation.

Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae), the causative agent of leprosy, parasitizes within the foamy or enlarged phagosome of macrophages where rich lipids accumulate. Although the mechanisms for lipid accumulation in the phagosome have been clarified, it is still unclear how such large amounts of lipids escape degradation. To further explore underlying mechanisms involved in lipid catabolism in M. leprae-infected host cells, we examined the expression of hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization and lipolysis, in human macrophage THP-1 cells. We found that infection by live M. leprae significantly suppressed HSL expression levels. This suppression was not observed with dead M. leprae or latex beads. Macrophage activation by peptidoglycan (PGN), the ligand for toll-like receptor 2 (TLR2), increased HSL expression; however, live M. leprae suppressed this increase. HSL expression was abolished in the slit-skin smear specimens from patients with lepromatous and borderline leprosy. In addition, the recovery of HSL expression was observed in patients who experienced a lepra reaction, which is a cell-mediated, delayed-type hypersensitivity immune response, or in patients who were successfully treated with multi-drug therapy. These results suggest that M. leprae suppresses lipid degradation through inhibition of HSL expression, and that the monitoring of HSL mRNA levels in slit-skin smear specimens may be a useful indicator of patient prognosis.

Analysis of Mycobacterium leprae gene expression using DNA microarray.

Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

Whole-genome tiling array analysis of Mycobacterium leprae RNA reveals high expression of pseudogenes and noncoding regions.

Whole-genome sequence analysis of Mycobacterium leprae has revealed a limited number of protein-coding genes, with half of the genome composed of pseudogenes and noncoding regions. We previously showed that some M. leprae pseudogenes are transcribed at high levels and that their expression levels change following infection. In order to clarify the RNA expression profile of the M. leprae genome, a tiling array in which overlapping 60-mer probes cover the entire 3.3-Mbp genome was designed. The array was hybridized with M. leprae RNA from the SHR/NCrj-rnu nude rat, and the results were compared to results from an open reading frame array and confirmed by reverse transcription-PCR. RNA expression was detected from genes, pseudogenes, and noncoding regions. The signal intensities obtained from noncoding regions were higher than those from pseudogenes. Expressed noncoding regions include the M. leprae unique repetitive sequence RLEP and other sequences without any homology to known functional noncoding RNAs. Although the biological functions of RNA transcribed from M. leprae pseudogenes and noncoding regions are not known, RNA expression analysis will provide insights into the bacteriological significance of the species. In addition, our study suggests that M. leprae will be a useful model organism for the study of the molecular mechanism underlying the creation of pseudogenes and the role of microRNAs derived from noncoding regions.

Pendrin is a novel autoantigen recognized by patients with autoimmune thyroid diseases.

CONTEXT: Pendrin is an apical protein of thyroid follicular cells, responsible for the efflux of iodide into the follicular lumen via an iodide-chloride transport mechanism. It is unknown whether pendrin is recognized by autoantibodies. OBJECTIVE: Our objective was to examine the prevalence of pendrin antibodies in autoimmune thyroid diseases and compare with that of thyroglobulin, thyroperoxidase, TSH receptor, and sodium iodide symporter antibodies. DESIGN: In a prevalent case-control study, we analyzed the sera of 140 autoimmune thyroid disease cases (100 with Graves' disease and 40 with Hashimoto's thyroiditis) and 80 controls (50 healthy subjects, 10 patients with papillary thyroid cancer, 10 with systemic lupus erythematosus, and 10 with rheumatoid arthritis). Pendrin antibodies were measured by immunoblotting using extract of COS-7 cells transfected with pendrin and a rabbit polyclonal pendrin antibody. RESULTS: Pendrin antibodies were found in 81% of the cases and 9% of controls (odds ratio = 44; P < 0.0001). Among cases, pendrin antibodies were more frequent and of higher titers in Hashimoto's thyroiditis than in Graves' disease. Pendrin antibodies correlated significantly with thyroglobulin, thyroperoxidase, and sodium iodide symporter antibodies but not with TSH receptor antibodies. Pendrin antibodies were equally effective as thyroglobulin and thyroperoxidase antibodies in diagnosis of autoimmune thyroid diseases, especially Hashimoto's thyroiditis. CONCLUSIONS: The study identifies pendrin as a novel autoantigen recognized by patients with autoimmune thyroid diseases and proposes the use of pendrin antibodies as an accurate diagnostic tool.

Expression of a thyroglobulin (Tg) variant in mouse kidney glomerulus.

Thyroglobulin (Tg) is an essential substrate for thyroid hormone biosynthesis whose production is primarily limited to the thyroid follicular cell. We have previously identified an approximately 1.2 kb fragment of Tg mRNA in cultured mouse mesangial cells, and in the present study provide evidence showing that this transcript is transcribed and translated into a unique protein (kTg) in the kidney, but not the thyroid gland. Cloning of kTg from a mouse kidney cDNA library showed that transcription starts in the middle of intron 41 of the Tg gene and continues in-frame with the remaining coding sequence of thyroid-derived Tg beginning with exon 42. Translation of this mRNA is predicted to yield a protein of 367 amino acids (40 kDa) containing a unique 13 amino acid sequence serving as a signal peptide followed by a 354 amino acid segment identical to the carboxy-terminal end of thyroid Tg. Western blot analysis with an antibody directed against the C-terminus of thyroid Tg detected a 40 kDa protein expressed in the kidney. Immunohistochemistry with this antibody showed that immunoreactive Tg was localized in podocytes and the mesangial area of the renal glomerulus. A part of a homologous transcript was also detected in human kidney, and the kTg protein was recognized by sera from Hashimoto's thyroiditis but not from controls. Together these results suggest that a unique low molecular weight variant of Tg is expressed in the kidney, where it could serve both physiological and pathological roles, including that of an autoantigen.

Detection of RNA expression from pseudogenes and non-coding genomic regions of Mycobacterium leprae.

We have previously reported that some pseudogenes are expressed in Mycobacterium leprae (M. leprae), the causative agent of leprosy, and that their expression levels alter upon infection of macrophages. We attempted to further examine the expression of pseudogene and non-coding genomic region in M. leprae, in this study. 19 Pseudogenes, 17 non-coding genomic regions, and 21 coding genes expression in M. leprae maintained in the footpads of the hypertensive nude rat (SHR/NCrj-rnu) were examined by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of some of these pseudogenes, non-coding genomic regions and coding genes were also examined in M. leprae from skin smear specimens obtained from patients with lepromatous leprosy by RT-PCR. Transcripts from pseudogenes, non-coding genomic regions and coding genes examined in this study were clearly observed in M. leprae. The expression patterns of some of these transcripts vary greatly among different leprosy patients. These results indicate that some of pseudogenes and non-coding genomic regions are transcribed in M. leprae and analysis of RNA expression patterns including pseudogene and non-coding genomic region in M. leprae may be useful in understanding the pathological states of infected patients.

[Detection of RNA expression on whole genome analysis of Mycobacterium leprae by tiling array].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. Although it was thought that pseudogenes and non-coding regions were silent and junk, our previous studies indicated that RNA expression was detected from these regions. To elucidate comprehensive RNA expression pattern on M. leprae whole genome, tiling array was designed and total RNA of M. leprae Thai-53 strain was analyzed. As a result, highly expressed regions were detected among not only the gene regions but also pseudogenes and non-coding regions. Since some of the RNA expression levels were modulated by MDT, evaluation of RNA expression pattern might be a good indicator for the treatment of leprosy.

[Comprehensive analysis of RNA expression of Mycobacterium leprae and clinical and biological significance].

Completion of Mycobacterium leprae genome sequence revealed that there are many pseudogenes and non-coding regions, but rather small numbers of protein-coding genes. This result indicates that M. leprae is a very unique organism, and this future is important to understand the biological nature and/or pathogenicity of M. leprae, which remain unclear. We attempted to find the biological nature of M. leprae by detecting the gene and pseudogene regions transcribed at high level. We detected the genomic regions including pseudogenes and demonstrated that six out of twelve high expression regions were pseudogenes. In addition, its transcription level was changed when M. leprae infects macrophage. RNA was detected from genes, pseudogenes and non-coding regions. The expression levels of these regions were different among patients and a part of them is disappeared just after treatment. These results suggested that RNA derived from pseudogene and non-coding region have some function concerning the infection and/or intracellular parasitism and that the analysis of pseudogene and non-coding region expression pattern of M. leprae is available as a criterion for therapeutic effect and disease type of leprosy, and a prognostic marker.

Improved Oxygen Supply to Multicellular Spheroids Using A Gas-permeable Plate and Embedded Hydrogel Beads.

Culture systems for three-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated two methods to enhance oxygen supply: (1) using a culture plate with a gas-permeable polydimethylsiloxane (PDMS) membrane on the bottom, and; (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids, comprised of 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads, did not develop a necrotic core for nine days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

Fibro-Adipogenic Remodeling of the Diaphragm in Obesity-Associated Respiratory Dysfunction.

Respiratory dysfunction is a common complication of obesity, conferring cardiovascular morbidity and increased mortality and often necessitating mechanical ventilatory support. While impaired lung expansion in the setting of increased adipose mass and reduced central response to hypercapnia have been implicated as pathophysiological drivers, the impact of obesity on respiratory muscles-in particular, the diaphragm-has not been investigated in detail. Here, we demonstrate that chronic high-fat diet (HFD) feeding impairs diaphragm muscle function, as assessed in vivo by ultrasonography and ex vivo by measurement of contractile force. During an HFD time course, progressive adipose tissue expansion and collagen deposition within the diaphragm parallel contractile deficits. Moreover, intradiaphragmatic fibro-adipogenic progenitors (FAPs) proliferate with long-term HFD feeding while giving rise to adipocytes and type I collagen-depositing fibroblasts. Thrombospondin 1 (THBS1), a circulating adipokine, increases with obesity and induces FAP proliferation. These findings suggest a novel role for FAP-mediated fibro-adipogenic diaphragm remodeling in obesity-associated respiratory dysfunction.

Expression of adipose differentiation-related protein (ADRP) and perilipin in macrophages infected with Mycobacterium leprae.

Mycobacterium leprae survives and replicates within a lipid droplet stored in the enlarged phagosome of histiocytes, a typical feature of lepromatous leprosy that is thought to be an important nutrient source for the bacillus. However, the underlying mechanisms by which lipids accumulate within phagosomes remain unclear. Recently, it was revealed that the lipid droplet-associated proteins, including ADRP and perilipin, play essential roles in lipid accumulation in adipocytes or macrophages. Therefore, we attempted to examine the role of these proteins in leprosy pathogenesis. ADRP and perilipin localized to the phagosomal membrane, which contains M. leprae in skin biopsy specimens of lepromatous leprosy. ADRP expression was transiently increased after phagocytosis in THP-1 cells. However, high levels of ADRP expression persisted only when live M. leprae, but not dead bacilli or latex beads, was added. Furthermore, although peptidoglycan, a Toll-like receptor 2 ligand, suppressed the expression levels of ADRP and perilipin, M. leprae infection inhibited this suppression. These results suggest that live M. leprae has the ability to actively induce and support ADRP/perilipin expression to facilitate the accumulation of lipids within the phagosome and to further maintain a suitable environment for the intracellular survival within the macrophage.

[Molecular mechanisms for intracellular parasitisation and exclusion in macrophage infected with Mycobacterium leprae].

It was previously demonstrated that TLR2 and CORO1A (TACO, Coronin 1, p57) localize phagosome membrane of macrophage. However, the functional relationship between TLR2 and CORO1A was not known. We show here that there is a functional counteraction between TLR2 and CORO1A.

Designing 3-D Adipospheres for Quantitative Metabolic Study.

Quantitative assessment of adipose mitochondrial activity is critical for better understanding of adipose tissue function in obesity and diabetes. While the two-dimensional (2-D) tissue culture method has been sufficient to discover key molecules that regulate adipocyte differentiation and function, the method is insufficient to determine the role of extracellular matrix (ECM) molecules and their modifiers, such as matrix metalloproteinases (MMPs), in regulating adipocyte function in three-dimensional (3-D) in vivo-like microenvironments. By using a 3-D hanging drop tissue culture system, we are able to produce scalable 3-D adipospheres that are suitable for quantitative metabolic study in 3-D microenvironment.

Membrane-Tethered Metalloproteinase Expressed by Vascular Smooth Muscle Cells Limits the Progression of Proliferative Atherosclerotic Lesions.

BACKGROUND: The MMP (matrix metalloproteinase) family plays diverse and critical roles in directing vascular wall remodeling in atherosclerosis. Unlike secreted-type MMPs, a member of the membrane-type MMP family, MT1-MMP (membrane-type 1 MMP; MMP14), mediates pericellular extracellular matrix degradation that is indispensable for maintaining physiological extracellular matrix homeostasis. However, given the premature mortality exhibited by MT1-MMP-null mice, the potential role of the proteinase in atherogenesis remains elusive. We sought to determine the effects of both MT1-MMP heterozygosity and tissue-specific gene targeting on atherogenesis in APOE (apolipoprotein E)-null mice. METHODS AND RESULTS: MT1-MMP heterozygosity in the APOE-null background (Mmp14+/-Apoe-/- ) significantly promoted atherogenesis relative to Mmp14+/+Apoe-/- mice. Furthermore, the tissue-specific deletion of MT1-MMP from vascular smooth muscle cells (VSMCs) in SM22α-Cre(+)Mmp14F/FApoe-/- (VSMC-knockout) mice likewise increased the severity of atherosclerotic lesions. Although VSMC-knockout mice also developed progressive atherosclerotic aneurysms in their iliac arteries, macrophage- and adipose-specific MT1-MMP-knockout mice did not display this sensitized phenotype. In VSMC-knockout mice, atherosclerotic lesions were populated by hyperproliferating VSMCs (smooth muscle actin- and Ki67-double-positive cells) that were characterized by a proinflammatory gene expression profile. Finally, MT1-MMP-null VSMCs cultured in a 3-dimensional spheroid model system designed to mimic in vivo-like cell-cell and cell-extracellular matrix interactions, likewise displayed markedly increased proliferative potential. CONCLUSIONS: MT1-MMP expressed by VSMCs plays a key role in limiting the progression of atherosclerosis in APOE-null mice by regulating proliferative responses and inhibiting the deterioration of VSMC function in atherogenic vascular walls.

Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells.

Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays an important role in endocytosis.

A Novel Role for Flotillin-Containing Lipid Rafts in Negative-Feedback Regulation of Thyroid-Specific Gene Expression by Thyroglobulin.

BACKGROUND: Thyroglobulin (Tg) stored in thyroid follicles regulates follicular function in thyroid hormone (TH) synthesis by suppressing thyroid-specific gene expression in a concentration-dependent manner. Thus, Tg is an intrinsic negative-feedback regulator that can restrain the effect of thyrotropin (TSH) in the follicle. However, the underlying mechanisms by which Tg exerts its prominent autoregulatory effect following recognition by thyrocytes remains unclear. METHODS: In order to identify potential proteins that recognize and interact with Tg, mass spectrometry was used to analyze immunoprecipitated Tg-bound proteins derived from Tg-treated rat thyroid FRTL-5 cells. RESULTS: Flotillin 1 and flotillin 2, two homologs that are integral membrane proteins in lipid rafts, were identified as novel Tg-binding proteins with high confidence. Further studies revealed that flotillins physically interact with endocytosed Tg, and together these proteins redistribute from the cell membrane to cytoplasmic vesicles. Treatment with the lipid raft disrupter methyl-ß-cyclodextrin abolished both the endocytosis and the negative-feedback effect of Tg on thyroid-specific gene expression. Meanwhile, siRNA-mediated knockdown of flotillin 1 or flotillin 2 also significantly inhibited Tg effects on gene expression. CONCLUSION: Together these results indicate that flotillin-containing lipid rafts are essential for follicular Tg to be recognized by thyrocytes and exert its negative-feedback effects in the thyroid.

The exocyst complex regulates free fatty acid uptake by adipocytes.

The exocyst is an octameric molecular complex that drives vesicle trafficking in adipocytes, a rate-limiting step in insulin-dependent glucose uptake. This study assessed the role of the exocyst complex in regulating free fatty acid (FFA) uptake by adipocytes. Upon differentiating into adipocytes, 3T3-L1 cells acquire the ability to incorporate extracellular FFAs in an insulin-dependent manner. A kinetic assay using fluoresceinated FFA (C12 dodecanoic acid) uptake allows the real-time monitoring of FFA internalization by adipocytes. The insulin-dependent uptake of C12 dodecanoic acid by 3T3-L1 adipocytes is mediated by Akt and phosphatidylinositol 3 (PI3)-kinase. Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes. Consistent with the roles played by Exo70 and Sec8 in FFA uptake, mCherry-tagged Exo70 and HA-tagged Sec8 partially colocalize with lipid droplets within adipocytes, suggesting their active roles in the development of lipid droplets. Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex. This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.

Leprosy as a model of immunity.

 Leprosy displays a spectrum of clinical manifestations, such as lepromatous and tuberculoid leprosy, and type I and II lepra reactions, which are thought to be a reflection of the host's immunological response against Mycobacterium leprae. Therefore, differential recognition of M. leprae, as well as its degraded components, and subsequent activation of cellular immunity will be an important factor for the clinical manifestation of leprosy. Although M. leprae mainly parasitizes tissue macrophages in the dermis and the Schwann cells of peripheral nerves, the presence of M. leprae in other organs, such as the liver, may also play important roles in the further modification of seesaw-like bipolar phenotypes of leprosy. Thus, leprosy is an exciting model for investigating the role of the human immune system in host defense and susceptibility to infection.