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1.
Nat Commun ; 12(1): 2265, 2021 04 15.
Article in English | MEDLINE | ID: mdl-33859199

ABSTRACT

Nerve-glia (NG2) glia or oligodendrocyte precursor cells (OPCs) are distributed throughout the gray and white matter and generate myelinating cells. OPCs in white matter proliferate more than those in gray matter in response to platelet-derived growth factor AA (PDGF AA), despite similar levels of its alpha receptor (PDGFRα) on their surface. Here we show that the type 1 integral membrane protein neuropilin-1 (Nrp1) is expressed not on OPCs but on amoeboid and activated microglia in white but not gray matter in an age- and activity-dependent manner. Microglia-specific deletion of Nrp1 compromised developmental OPC proliferation in white matter as well as OPC expansion and subsequent myelin repair after acute demyelination. Exogenous Nrp1 increased PDGF AA-induced OPC proliferation and PDGFRα phosphorylation on dissociated OPCs, most prominently in the presence of suboptimum concentrations of PDGF AA. These findings uncover a mechanism of regulating oligodendrocyte lineage cell density that involves trans-activation of PDGFRα on OPCs via Nrp1 expressed by adjacent microglia.


Subject(s)
Demyelinating Diseases/pathology , Microglia/physiology , Neuropilin-1/metabolism , Oligodendrocyte Precursor Cells/physiology , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Remyelination , Animals , Cell Differentiation , Cell Lineage , Cell Proliferation , Cells, Cultured , Cerebellum/cytology , Cerebellum/growth & development , Corpus Callosum/cytology , Corpus Callosum/drug effects , Corpus Callosum/growth & development , Corpus Callosum/pathology , Demyelinating Diseases/chemically induced , Disease Models, Animal , Female , Humans , Lysophosphatidylcholines/administration & dosage , Lysophosphatidylcholines/toxicity , Male , Mice , Mice, Transgenic , Microglia/drug effects , Microglia/ultrastructure , Microscopy, Electron, Transmission , Models, Animal , Myelin Sheath/metabolism , Neuropilin-1/genetics , Oligodendroglia/physiology , Platelet-Derived Growth Factor/metabolism , Primary Cell Culture
2.
Sci Rep ; 11(1): 3552, 2021 02 11.
Article in English | MEDLINE | ID: mdl-33574458

ABSTRACT

Oligodendrocyte precursor cells (NG2 glia) are uniformly distributed proliferative cells in the mammalian central nervous system and generate myelinating oligodendrocytes throughout life. A subpopulation of OPCs in the neocortex arises from progenitor cells in the embryonic ganglionic eminences that also produce inhibitory neurons. The neuronal fate of some progenitor cells is sealed before birth as they become committed to the oligodendrocyte lineage, marked by sustained expression of the oligodendrocyte transcription factor Olig2, which represses the interneuron transcription factor Dlx2. Here we show that misexpression of Dlx2 alone in postnatal mouse OPCs caused them to switch their fate to GABAergic neurons within 2 days by downregulating Olig2 and upregulating a network of inhibitory neuron transcripts. After two weeks, some OPC-derived neurons generated trains of action potentials and formed clusters of GABAergic synaptic proteins. Our study revealed that the developmental molecular logic can be applied to promote neuronal reprogramming from OPCs.


Subject(s)
Embryonic Development/genetics , GABAergic Neurons/metabolism , Homeodomain Proteins/genetics , Oligodendrocyte Precursor Cells/metabolism , Oligodendrocyte Transcription Factor 2/genetics , Transcription Factors/genetics , Cell Proliferation/genetics , Cellular Reprogramming/genetics , Central Nervous System , Gene Expression Regulation/genetics , Homeodomain Proteins/metabolism , Neuroglia/metabolism , Synapses/genetics , Transcription Factors/metabolism
3.
Glia ; 69(3): 792-811, 2021 03.
Article in English | MEDLINE | ID: mdl-33098183

ABSTRACT

Oligodendrocyte precursor cells (OPCs), also known as NG2 cells or polydendrocytes, are distributed widely throughout the developing and mature central nervous system. They remain proliferative throughout life and are an important source of myelinating cells in normal and demyelinating brain as well as a source of glioma, the most common type of primary brain tumor with a poor prognosis. OPC proliferation is dependent on signaling mediated by platelet-derived growth factor (PDGF) AA binding to its alpha receptor (PDGFRα). Here, we describe a group of structurally related compounds characterized by the presence of a basic guanidine group appended to an aromatic core that is effective in specifically repressing the transcription of Pdgfra but not the related beta receptor (Pdgfrb) in OPCs. These compounds specifically and dramatically reduced proliferation of OPCs but not that of astrocytes and did not affect signal transduction by PDGFRα. These findings suggest that the compounds could be further developed for potential use in combinatorial treatment strategies for neoplasms with dysregulated PDGFRα function.


Subject(s)
Oligodendrocyte Precursor Cells , Cell Proliferation , Guanidine , Oligodendroglia , Receptor, Platelet-Derived Growth Factor alpha/genetics
4.
Dev Biol ; 468(1-2): 133-145, 2020 12 01.
Article in English | MEDLINE | ID: mdl-32768399

ABSTRACT

The importance of skeletal muscle for rib development and patterning in the mouse embryo has not been resolved, largely because different experimental approaches have yielded disparate results. In this study, we utilize both gene knockouts and muscle cell ablation approaches to re-visit the extent to which rib growth and patterning are dependent on developing musculature. Consistent with previous studies, we show that rib formation is highly dependent on the MYOD family of myogenic regulatory factors (MRFs), and demonstrate that the extent of rib formation is gene-, allele-, and dosage-dependent. In the absence of Myf5 and MyoD, one allele of Mrf4 is sufficient for extensive rib growth, although patterning is abnormal. Under conditions of limiting MRF dosage, MyoD is identified as a positive regulator of rib patterning, presumably due to improved intercostal muscle development. In contrast to previous muscle ablation studies, we show that diphtheria toxin subunit A (DTA)-mediated ablation of muscle progenitors or differentiated muscle, using MyoDiCre or HSA-Cre drivers, respectively, profoundly disrupts rib development. Further, a comparison of three independently derived Rosa26-based DTA knockin alleles demonstrates that the degree of rib perturbations in MyoDiCre/+/DTA embryos is markedly dependent on the DTA allele used, and may in part explain discrepancies with previous findings. The results support the conclusion that the extent and quality of rib formation is largely dependent on the dosage of Myf5 and Mrf4, and that both early myotome-sclerotome interactions, as well as later muscle-rib interactions, are important for proper rib growth and patterning.


Subject(s)
Body Patterning , Muscle, Skeletal/embryology , Ribs/embryology , Alleles , Animals , Gonadotropin-Releasing Hormone/analogs & derivatives , Mice, Transgenic , MyoD Protein/genetics , MyoD Protein/metabolism , Myogenic Regulatory Factor 5/genetics , Myogenic Regulatory Factor 5/metabolism , Myogenic Regulatory Factors/genetics , Myogenic Regulatory Factors/metabolism
5.
Glia ; 66(12): 2684-2699, 2018 12.
Article in English | MEDLINE | ID: mdl-30306660

ABSTRACT

NG2 is a type 1 integral membrane glycoprotein encoded by the Cspg4 gene. It is expressed on glial progenitor cells known as NG2 glial cells or oligodendrocyte precursor cells that exist widely throughout the developing and mature central nervous system and vascular mural cells but not on mature oligodendrocytes, astrocytes, microglia, neurons, or neural stem cells. Hence NG2 is widely used as a marker for NG2 glia in the rodent and human. The regulatory elements of the mouse Cspg4 gene and its flanking sequences have been used successfully to target reporter and Cre recombinase to NG2 glia in transgenic mice when used in a large 200 kb bacterial artificial chromosome cassette containing the 38 kb Cspg4 gene in the center. Despite the tightly regulated cell type- and stage-specific expression of NG2 in the brain and spinal cord, the mechanisms that regulate its transcription have remained unknown. Here, we describe a 1.45 kb intronic enhancer of the mouse Cspg4 gene that directed transcription of EGFP reporter to NG2 glia but not to pericytes in vitro and in transgenic mice. The 1.45 kb enhancer contained binding sites for SoxE and basic helix-loop-helix transcription factors, and its enhancer activity was augmented cooperatively by these factors, whose respective binding elements were found in close proximity to each other. Mutations in these binding elements abrogated the enhancer activity when tested in the postnatal mouse brain.


Subject(s)
Antigens/genetics , Antigens/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Developmental/genetics , Neuroglia/metabolism , Proteoglycans/genetics , Proteoglycans/metabolism , Age Factors , Animals , Animals, Newborn , Basic Helix-Loop-Helix Transcription Factors/genetics , Binding Sites/genetics , Brain/cytology , Chromatin Immunoprecipitation , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Histones/metabolism , Mice , Mice, Inbred ICR , Mice, Transgenic , Mutation/genetics , Oligodendrocyte Transcription Factor 2/genetics , Oligodendrocyte Transcription Factor 2/metabolism , SOXE Transcription Factors/genetics , SOXE Transcription Factors/metabolism , Transfection
6.
J Neurosci ; 38(9): 2359-2371, 2018 02 28.
Article in English | MEDLINE | ID: mdl-29382710

ABSTRACT

NG2 cells are a resident glial progenitor cell population that is uniformly distributed throughout the developing and mature mammalian CNS. Those in the postnatal CNS generate exclusively myelinating and non-myelinating oligodendrocytes and are thus equated with oligodendrocyte precursor cells. Prenatally, NG2 cells in the ventral gray matter of the forebrain generate protoplasmic astrocytes as well as oligodendrocytes. The fate conversion from NG2 cells into protoplasmic astrocytes is dependent on downregulation of the key oligodendrocyte transcription factor Olig2. We showed previously that constitutive deletion of Olig2 in NG2 cells converts NG2 cells in the neocortex into protoplasmic astrocytes at the expense of oligodendrocytes. In this study, we show that postnatal deletion of Olig2 caused NG2 cells in the neocortex but not in other gray matter regions to become protoplasmic astrocytes. However, NG2 cells in the neocortex became more resistant to astrocyte fate switch over the first 3 postnatal weeks. Fewer NG2 cells differentiated into astrocytes and did so with longer latency after Olig2 deletion at postnatal day 18 (P18) compared with deletion at P2. The high-mobility group transcription factor Sox10 was not downregulated for at least 1 month after Olig2 deletion at P18 despite an early transient upregulation of the astrocyte transcription factor NFIA. Furthermore, inhibiting cell proliferation in slice culture reduced astrocyte differentiation from Olig2-deleted perinatal NG2 cells, suggesting that cell division might facilitate nuclear reorganization needed for astrocyte transformation.SIGNIFICANCE STATEMENT NG2 cells are glial progenitor cells that retain a certain degree of lineage plasticity. In the normal postnatal neocortex, they generate mostly oligodendrocyte lineage cells. When the oligodendrocyte transcription factor Olig2 is deleted in NG2 cells in the neocortex, they switch their fate to protoplasmic astrocytes. However, the efficiency of the fate switch decreases with age over the first 3 postnatal weeks and is reduced when cell proliferation is inhibited. As the neocortex matures, sustained expression of the oligodendrocyte lineage-specific key transcription factor Sox10 becomes less dependent on Olig2. Together, our findings suggest a gradual stabilization of the oligodendrocyte lineage genes and loss of lineage plasticity during the first 3 weeks after birth, possibly due to nuclear reorganization.


Subject(s)
Astrocytes/cytology , Cell Differentiation/physiology , Neural Stem Cells/cytology , Neurogenesis/physiology , Oligodendrocyte Transcription Factor 2/metabolism , Animals , Cell Lineage , Mice , Mice, Knockout , Neocortex/cytology , Oligodendroglia/cytology
7.
PLoS One ; 10(8): e0134901, 2015.
Article in English | MEDLINE | ID: mdl-26263379

ABSTRACT

NF-κB signaling plays an important role in tumor cell proliferation, cell survival, angiogenesis, invasion, metastasis and drug/radiation resistance. Combination therapy involving NF-κB pathway inhibition is an attractive strategy for the treatment of advanced forms of thyroid cancer. This study was designed to test the efficacy of NF-κB pathway inhibition in combination with cytotoxic chemotherapy, using docetaxel and ionizing radiation in in vitro models of thyroid cancer. We found that while both docetaxel and ionizing radiation activated NF-κB signaling in thyroid cancer cells, there was no synergistic effect on cell proliferation and/or programmed cell death with either genetic (transduction of a dominant negative mutant form of IκBα) or pharmacologic (proteasome inhibitor bortezomib and IKKß inhibitor GO-Y030) inhibition of the NF-κB pathway in thyroid cancer cell lines BCPAP, 8505C, THJ16T and SW1736. Docetaxel plus bortezomib synergistically decreased in vitro invasion of 8505C cells, but not in the other cell lines. Screening of a panel of clinically relevant targeted therapies for synergy with genetic NF-κB inhibition in a proliferation/cytotoxicity assay identified the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) as a potential candidate. However, the synergistic effect was confirmed only in the BCPAP cells. These results indicate that NF-κB inhibitors are unlikely to be beneficial as combination therapy with taxane cytotoxic chemotherapy, external radiation therapy or radioiodine therapy. There may be unique circumstances where NF-κB inhibitors may be considered in combination with docetaxel to reduce tumor invasion or in combination with HDAC inhibitors to reduce tumor growth, but this does not appear to be a combination therapy that could be broadly applied to patients with advanced thyroid cancer. Further research may identify which subsets of patients/tumors may respond to this therapeutic approach.


Subject(s)
Antineoplastic Agents/pharmacology , NF-kappa B/metabolism , Signal Transduction/drug effects , Thyroid Neoplasms/metabolism , Antineoplastic Agents/therapeutic use , Bortezomib/pharmacology , Caspase 3/metabolism , Caspase 7/metabolism , Cell Death/drug effects , Cell Death/radiation effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Combined Modality Therapy , Docetaxel , Humans , Inhibitory Concentration 50 , Molecular Targeted Therapy , Neoplasm Staging , Radiation, Ionizing , Signal Transduction/radiation effects , Taxoids/pharmacology , Thyroid Neoplasms/pathology , Thyroid Neoplasms/therapy
8.
Front Aging Neurosci ; 7: 62, 2015.
Article in English | MEDLINE | ID: mdl-26042028

ABSTRACT

Pax7 expressing muscle stem cells accompany all skeletal muscles in the body and in healthy individuals, efficiently repair muscle after injury. Currently, the in vitro manipulation and culture of these cells is still in its infancy, yet muscle stem cells may be the most promising route toward the therapy of muscle diseases such as muscular dystrophies. It is often overlooked that muscular dystrophies affect head and body skeletal muscle differently. Moreover, these muscles develop differently. Specifically, head muscle and its stem cells develop from the non-somitic head mesoderm which also has cardiac competence. To which extent head muscle stem cells retain properties of the early head mesoderm and might even be able to switch between a skeletal muscle and cardiac fate is not known. This is due to the fact that the timing and mechanisms underlying head muscle stem cell development are still obscure. Consequently, it is not clear at which time point one should compare the properties of head mesodermal cells and head muscle stem cells. To shed light on this, we traced the emergence of head muscle stem cells in the key vertebrate models for myogenesis, chicken, mouse, frog and zebrafish, using Pax7 as key marker. Our study reveals a common theme of head muscle stem cell development that is quite different from the trunk. Unlike trunk muscle stem cells, head muscle stem cells do not have a previous history of Pax7 expression, instead Pax7 expression emerges de-novo. The cells develop late, and well after the head mesoderm has committed to myogenesis. We propose that this unique mechanism of muscle stem cell development is a legacy of the evolutionary history of the chordate head mesoderm.

9.
J Clin Endocrinol Metab ; 99(8): E1436-44, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24758177

ABSTRACT

CONTEXT: Development of novel strategies in the treatment of advanced thyroid cancer are needed. Our laboratory has previously identified a role for nuclear factor κB (NF-κB) signaling in human thyroid cancer cell growth, survival, and invasion. OBJECTIVE: Our goal was to establish the role of NF-κB signaling on thyroid cancer growth and metastases in vivo and to begin to dissect mechanisms regulating this effect. SETTING AND DESIGN: We examined tumor formation of five thyroid cancer cell lines in an in vivo model of thyroid cancer and observed tumor establishment in two of the cell lines (8505C and BCPAP). RESULTS: Inhibition of NF-κB signaling by overexpression of a dominant-negative IκBα (mIκBα) significantly inhibited thyroid tumor growth in tumors derived from both cell lines. Further studies in an experimental metastasis model demonstrated that NF-κB inhibition impaired growth of tumor metastasis and prolonged mouse survival. Proliferation (mitotic index) was decreased in 8505C tumors, but not in BCPAP tumors, while in vitro angiogenesis and in vivo tumor vascularity were significantly inhibited by mIkBα only in the BCPAP cells. Cytokine antibody array analysis demonstrated that IL-8 secretion was blocked by mIκBα expression. Interestingly, basal NF-κB activity and IL-8 levels were significantly higher in the two tumorigenic cell lines compared with the nontumorigenic lines. Furthermore, IL-8 transcript levels were elevated in high-risk human tumors, suggesting that NF-κB and IL-8 are associated with more aggressive tumor behavior. CONCLUSIONS: These studies suggest that NF-κB signaling is a key regulator of angiogenesis and growth of primary and metastatic thyroid cancer, and that IL-8 may be an important downstream mediator of NF-κB signaling in advanced thyroid cancer growth and progression.


Subject(s)
Interleukin-8/metabolism , NF-kappa B/physiology , Neovascularization, Pathologic/genetics , Thyroid Neoplasms/blood supply , Thyroid Neoplasms/pathology , Animals , Cells, Cultured , Gene Expression Regulation, Neoplastic/drug effects , Human Umbilical Vein Endothelial Cells , Humans , I-kappa B Proteins/genetics , Male , Mice , Mice, Nude , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , Neoplasm Metastasis , Neoplasm Transplantation , Thyroid Neoplasms/genetics
10.
Mol Cancer ; 13: 62, 2014 Mar 19.
Article in English | MEDLINE | ID: mdl-24645981

ABSTRACT

BACKGROUND: Thyroid cancer is the most common endocrine malignancy, and many patients with metastatic differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) fail to respond to conventional therapies, resulting in morbidity and mortality. Additional therapeutic targets and treatment options are needed for these patients. We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ATC and confers an aggressive phenotype when overexpressed in DTC cells. METHODS: Microarray analysis was used to identify downstream targets of PPARγ in ATC cells. Western blot analysis and immunohistochemistry (IHC) were used to assess thioredoxin interacting protein (TXNIP) expression in thyroid cancer cell lines and primary tumor specimens. Retroviral transduction was used to generate ATC cell lines that overexpress TXNIP, and assays that assess glucose uptake, viable cell proliferation, and invasion were used to characterize the in vitro properties of these cells. An orthotopic thyroid cancer mouse model was used to assess the effect of TXNIP overexpression in ATC cell lines in vivo. RESULTS: Using microarray analysis, we show that TXNIP is highly upregulated when PPARγ is depleted from ATC cells. Using Western blot analysis and IHC, we show that DTC and ATC cells exhibit differential TXNIP expression patterns. DTC cell lines and patient tumors have high TXNIP expression in contrast to low or absent expression in ATC cell lines and tumors. Overexpression of TXNIP decreases the growth of HTh74 cells compared to vector controls and inhibits glucose uptake in the ATC cell lines HTh74 and T238. Importantly, TXNIP overexpression in T238 cells results in attenuated tumor growth and decreased metastasis in an orthotopic thyroid cancer mouse model. CONCLUSIONS: Our findings indicate that TXNIP functions as a tumor suppressor in thyroid cells, and its downregulation is likely important in the transition from differentiated to advanced thyroid cancer. These studies underscore the potential of TXNIP as a novel therapeutic target and prognostic indicator in advanced thyroid cancer.


Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Genes, Tumor Suppressor/physiology , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Heterografts , Humans , Immunohistochemistry , Mice , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic
11.
Dev Biol ; 384(1): 114-27, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24055173

ABSTRACT

Skeletal myogenesis in the embryo is regulated by the coordinated expression of the MyoD family of muscle regulatory factors (MRFs). MyoD and Myf-5, which are the primary muscle lineage-determining factors, function in a partially redundant manner to establish muscle progenitor cell identity. Previous diphtheria toxin (DTA)-mediated ablation studies showed that MyoD+ progenitors rescue myogenesis in embryos in which Myf-5-expressing cells were targeted for ablation, raising the possibility that the regulative behavior of distinct, MRF-expressing populations explains the functional compensatory activities of these MRFs. Using MyoD(iCre) mice, we show that DTA-mediated ablation of MyoD-expressing cells results in the cessation of myogenesis by embryonic day 12.5 (E12.5), as assayed by myosin heavy chain (MyHC) and Myogenin staining. Importantly, MyoD(iCre/+);R26(DTA/+) embryos exhibited a concomitant loss of Myf-5+ progenitors, indicating that the vast majority of Myf-5+ progenitors express MyoD, a conclusion consistent with immunofluorescence analysis of Myf-5 protein expression in MyoD(iCre) lineage-labeled embryos. Surprisingly, staining for the paired box transcription factor, Pax7, which functions genetically upstream of MyoD in the trunk and is a marker for fetal myoblasts and satellite cell progenitors, was also lost by E12.5. Specific ablation of differentiating skeletal muscle in ACTA1Cre;R26(DTA/+) embryos resulted in comparatively minor effects on MyoD+, Myf-5+ and Pax7+ progenitors, indicating that cell non-autonomous effects are unlikely to explain the rapid loss of myogenic progenitors in MyoD(iCre/+);R26(DTA/+) embryos. We conclude that the vast majority of myogenic cells transit through a MyoD+ state, and that MyoD+ progenitors are essential for myogenesis and stem cell development.


Subject(s)
Muscle, Skeletal/embryology , MyoD Protein/genetics , Satellite Cells, Skeletal Muscle/metabolism , Stem Cells/metabolism , Animals , Cell Differentiation , Embryo, Mammalian/metabolism , Mice , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , MyoD Protein/metabolism
12.
PPAR Res ; 2011: 171765, 2011.
Article in English | MEDLINE | ID: mdl-22194735

ABSTRACT

Undifferentiated (anaplastic) thyroid cancer (ATC) is one of the most aggressive human malignancies and no effective therapy is currently available. We show here that PPARγ levels are elevated in cells derived from ATC. Depletion of PPARγ in HTh74 ATC cells resulted in decreased cell growth, cell cycle arrest and a reduction in pRb and cyclin A and B1 levels. We further showed that both flank and orthotopic thyroid tumors derived from PPARγ-depleted cells grew more slowly than PPARγ-expressing cells. When PPARγ was overexpressed in more differentiated thyroid cancer BCPAP cells which lack PPARγ, there was increased growth and raised pRb and cyclin A and B1 levels. Finally, PPARγ depletion in ATC cells decreased their invasive capacity whereas overexpression in PTC cells increased invasiveness. These data suggest that PPARγ may play a detrimental role in thyroid cancer and that targeting it therapeutically may lead to improved treatment of advanced thyroid cancer.

13.
Mol Cell Endocrinol ; 339(1-2): 1-6, 2011 Jun 06.
Article in English | MEDLINE | ID: mdl-21458528

ABSTRACT

Retinoid X receptor (RXR) signaling influences thyrotrope function. Synthetic RXR agonists, rexinoids, can cause central hypothyroidism. To test the hypothesis that endogenous rexinoids contribute to the TSH 'set point', TαT1 mouse thyrotrope cells were treated with a rexinoid antagonist, LG101208. Increasing concentrations of LG101208 significantly increased TSHß mRNA levels, indicating that the rexinoid antagonist may interfere with RXR-signaling by an endogenous rexinoid in thyrotropes. When the same experiments were repeated in the presence of charcoal-stripped serum the effect of the rexinoid antagonist was lost. Pretreatment with the transcription inhibitor DRB blocked the increase of TSHß mRNA levels by rexinoid antagonist, indicating the primary effect is at the level of gene transcription. Mice treated with LG101208 had higher levels of serum T4, T4/TSH ratios as well as pituitary α-subunit and TSHß mRNA compared with vehicle treated mice. Hypothalamic TRH levels were unchanged. In summary, the rexinoid antagonist, LG101208, increases TSH subunit mRNA levels in thyrotrope cells and mouse pituitaries, primarily at the level of gene transcription. These data suggest that an "endogenous rexinoid" contributes to the TSH 'set point' in thyrotropes.


Subject(s)
Hypothalamus/metabolism , Pituitary Gland/metabolism , Retinoid X Receptors/antagonists & inhibitors , Retinoids/pharmacology , Thyroid Gland/metabolism , Animals , Cell Line, Tumor , Glycoprotein Hormones, alpha Subunit/blood , Glycoprotein Hormones, alpha Subunit/genetics , Glycoprotein Hormones, alpha Subunit/metabolism , Hypothalamus/drug effects , Male , Mice , Mice, 129 Strain , Pituitary Gland/drug effects , Thyroid Gland/drug effects , Thyrotropin, beta Subunit/blood , Thyrotropin, beta Subunit/genetics , Thyrotropin, beta Subunit/metabolism , Thyroxine/blood , Transcription, Genetic/drug effects
14.
BMC Biol ; 8: 113, 2010 Aug 31.
Article in English | MEDLINE | ID: mdl-20807406

ABSTRACT

BACKGROUND: Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development. RESULTS: We examined the expression of key testicular (SRY, SOX9, AMH and FGF9) and ovarian (WNT4, RSPO1, FOXL2 and FST) markers during gonadal development in the marsupial tammar wallaby (Macropus eugenii) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of AMH and SOX9 as in the mouse and human testis, but this upregulation was initiated before the peak in SRY expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of FOXL2 and WNT4, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of SRY, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad. CONCLUSIONS: We have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein. See commentary: http://www.biomedcentral.com/1741-7007/8/110.


Subject(s)
Estrogens/metabolism , Gonads/growth & development , Gonads/metabolism , Macropodidae/growth & development , SOX9 Transcription Factor/metabolism , Animals , Female , Macropodidae/metabolism , Male , Ovary/growth & development , Ovary/metabolism , Testis/growth & development , Testis/metabolism
15.
Pituitary ; 9(1): 11-8, 2006.
Article in English | MEDLINE | ID: mdl-16703404

ABSTRACT

Glycoprotein-hormone alpha-subunit deficient (alphaSUnull) mice are hypothyroid and hypogonadal due to the absence of functional TSH, LH and FSH, despite normal production of the corresponding beta subunits. Pituitary tumors spontaneously developing in alphaSUnull mice were propagated in hypothyroid mice. The purpose of the current studies was to compare the gene expression profile of these alphaSUnull tumors with previously characterized TtT-97 thyrotropic tumors. A group of animals bearing each tumor type was treated with thyroid hormone (T4) prior to tumor removal. Both tumor types equally expressed TSHbeta mRNA, which significantly decreased when exposed to T4, whereas alpha-subunit mRNA was absent in alphaSUnull tumors. Northern blot analysis was performed using cDNA probes for the following transcription factors: Pit1, GATA2, pLIM, Msx1, Ptx1 and Ptx2. Both tumors were found to contain identical transcripts with similar responses to T4, with the exception of Pit1. In contrast to the signal pattern seen in TtT-97, only two bands were seen in alphaSUnull tumors, which were similar in size to those in alphaTSH cells, a thyrotropic cell line that lacks TSHbeta-subunit expression and Pit1 protein. However, western blot analysis revealed a protein band in the alphaSUnull tumors consistent with Pit1, while this signal was absent in alphaTSH cells. Northern blot analysis was also performed with specific cDNA probes for the following receptors: TRbeta1, TRbeta2, TRalpha1, non-T3 binding alpha2, RXRgamma and Sst5. Similarly-sized transcripts were found in both types of tumor, although the signal for Sst5 was seen in T4-treated alphaSUnull tumors only with a more sensitive RT-PCR analysis. The overall similarity between the two tumor types renders the alphaSUnull tumor as a suitable thyrotropic tumor model.


Subject(s)
Glycoprotein Hormones, alpha Subunit/deficiency , Pituitary Neoplasms/etiology , Pituitary Neoplasms/genetics , Thyrotropin, beta Subunit/genetics , Transcription Factors/metabolism , Animals , DNA, Complementary/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Thyrotropin/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics
16.
Mol Endocrinol ; 20(6): 1366-77, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16543408

ABSTRACT

GATA2 is expressed in the pituitary during development and in adult gonadotropes and thyrotropes. It is proposed to be important for gonadotrope and thyrotrope cell fate choice and for TSH production. To test this idea, we produced a pituitary-specific knockout of Gata2, designed so that the DNA-binding zinc-finger region is deleted in the presence of a pituitary-specific recombinase transgene. These mice have reduced secretion of gonadotropins basally and in response to castration challenge, although the mice are fertile. GATA2 deficiency also compromises thyrotrope function. Mutants have fewer thyrotrope cells at birth, male Gata2-deficient mice exhibit growth delay from 3-9 wk of age, and adult mutants produce less TSH in response to severe hypothyroidism after radiothyroidectomy. Therefore, Gata2 appears to be dispensable for gonadotrope and thyrotrope cell fate and maintenance, but important for optimal gonadotrope and thyrotrope function. Gata2-deficient mice exhibit elevated levels of Gata3 transcripts in the pituitary gland, suggesting that GATA3 can compensate for GATA2.


Subject(s)
GATA2 Transcription Factor/deficiency , Pituitary Gland/physiopathology , Animals , Animals, Newborn , Base Sequence , Body Weight , DNA/genetics , Female , Follicle Stimulating Hormone/blood , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/physiology , GATA3 Transcription Factor/genetics , Gonadotropins, Pituitary/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Orchiectomy , Pituitary Gland/pathology , Pregnancy , Thyroidectomy , Thyrotropin/biosynthesis , Transcription, Genetic
17.
Mol Cell Biol ; 26(5): 1722-30, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16478993

ABSTRACT

The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.


Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Receptors, Progesterone/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Cloning, Molecular , Cytoplasm/metabolism , HSP90 Heat-Shock Proteins/genetics , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Molecular Chaperones/metabolism , Molecular Sequence Data , Protein Structure, Tertiary , Receptors, Progesterone/genetics , Signal Transduction , Tacrolimus Binding Proteins/metabolism
18.
Mol Endocrinol ; 20(5): 1073-89, 2006 May.
Article in English | MEDLINE | ID: mdl-16396960

ABSTRACT

Mediator (MED) 220/thyroid receptor-associated protein (TRAP) 220 is a transcriptional mediator that interacts with liganded thyroid/steroid hormone receptors. MED220 haploinsufficient heterozygotes exhibited hypothyroidism and reduced TSHbeta transcripts, suggesting a specific function for TSHbeta transcription. We previously demonstrated that Pit-1 and GATA-2 can bind to a composite element within the proximal TSHbeta promoter and synergistically activate transcription. We detected MED220 expression in TtT-97 thyrotropes by Northern and Western blot analysis. Cotransfections in CV-1 cells showed that Pit-1, GATA-2, or MED220 alone did not markedly stimulate the TSHbeta promoter. However, Pit-1 plus GATA-2 resulted in an 10-fold activation, demonstrating synergistic cooperativity. Titration of MED220 resulted in a further dose-dependent stimulation up to 25-fold that was promoter specific. Glutathione-S-transferase interaction studies showed that MED220 or GATA-2 each bound the homeodomain of Pit-1, whereas MED220 interacted independently with each zinc finger of GATA-2 but not with either terminus. MED220 interacted with GATA-2 and Pit-1 over a broad region of its N terminus. These regions of interaction were also important for maximal function. Coimmunoprecipitation confirmed that all three factors can interact in thyrotropes and chromatin immunoprecipitation demonstrated in vivo occupancy on the proximal TSHbeta promoter. Thus, the TSHbeta gene is maximally activated by a combination of three thyrotrope transcription factors that act via both protein-DNA and protein-protein interactions.


Subject(s)
GATA2 Transcription Factor/metabolism , Gene Expression Regulation , Thyrotropin, beta Subunit/genetics , Trans-Activators/metabolism , Transcription Factor Pit-1/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Animals , GATA2 Transcription Factor/genetics , Mediator Complex Subunit 1 , Mice , Neoplasms/genetics , Neoplasms/metabolism , Promoter Regions, Genetic , Protein Interaction Mapping , Protein Structure, Tertiary , Sequence Deletion , Thyrotropin/metabolism , Transcription Factor Pit-1/genetics , Transcription Factors/genetics
19.
Endocrinology ; 147(1): 272-82, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16223861

ABSTRACT

In this report we have examined changes in cell growth parameters, cell cycle effectors, and signaling pathways that accompany thyrotrope growth arrest by thyroid hormone (TH) and growth resumption after its withdrawal. Flow cytometry and immunohistochemistry of proliferation markers demonstrated that TH treatment of thyrotrope tumors resulted in a reduction in the fraction of cells in S-phase that is restored upon TH withdrawal. This is accompanied by dephosphorylation and rephosphorylation of retinoblastoma (Rb) protein. The expression levels of cyclin-dependent kinase 2 and cyclin A, as well as cyclin-dependent kinase 1 and cyclin B, were decreased by TH, and after withdrawal not only did these regulators of Rb phosphorylation and mitosis increase in their expression but so too did the D1 and D3 cyclins. We also noted a rapid induction and subsequent disappearance of the type 5 receptor for the growth inhibitor somatostatin with TH treatment and withdrawal, respectively. Because somatostatin can arrest growth by activating MAPK pathways, we examined these pathways in TtT-97 tumors and found that the ERK pathway and several of its upstream and downstream effectors, including cAMP response element binding protein, were activated with TH treatment and deactivated after its withdrawal. This led to the hypothesis that TH, acting through increased type 5 somatostatin receptor, could activate the ERK pathway leading to cAMP response element binding protein-dependent decreased expression of critical cell cycle proteins, specifically cyclin A, resulting in hypophosphorylation of Rb and its subsequent arrest of S-phase progression. These processes are reversed when TH is withdrawn, resulting in an increase in the fraction of S-phase cells.


Subject(s)
Thyroid Gland/cytology , Thyroid Hormones/pharmacology , Animals , Disease Models, Animal , Hypothyroidism/pathology , Mice , Mice, Inbred Strains , Mitogen-Activated Protein Kinase Kinases/metabolism , Proliferating Cell Nuclear Antigen/analysis , Thyroid Gland/drug effects , Thyroidectomy , Thyrotropin/genetics , Thyrotropin/pharmacology
20.
Endocrinology ; 147(3): 1438-51, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16306084

ABSTRACT

Retinoid X receptor (RXR)-selective retinoids (rexinoids) can cause central hypothyroidism in humans, and this effect has been confirmed in rodent models. In this report, we characterized the effect of rexinoids on the hypothalamic-pituitary-thyroid axis in mice and TSH regulation in a thyrotrope-derived cell line. The synthetic rexinoid (LG 268) suppressed TSH and T4 levels in mice. Hypothalamic TRH mRNA was unaffected, but steady-state pituitary TSHbeta mRNA levels were significantly lowered, suggesting a direct effect of rexinoids on thyrotropes. LG 268 suppressed TSH protein secretion and TSHbeta mRNA in TalphaT1 thyrotropes as early as 8 h after treatment, whereas the retinoic acid receptor-selective retinoid (TTNPB) had no effect. Type 2 iodothyronine deiodinase (D2) mRNA and activity were suppressed by LG 268 in TalphaT1 cells, whereas only D2 mRNA was suppressed in mouse pituitaries. LG 268 suppressed TSHbeta promoter activity by 42% and the -200 to -149 region accounted for a majority of the LG 268-mediated suppression of promoter activity. The RXRgamma isotype is expressed in thyrotropes. In vitro transfection and in vivo transgenic studies indicate that any RXR isotype can mediate TSH suppression by rexinoids, but the RXRgamma isotype is most efficient at mediating this response. RXRgamma-deficient mice lacked pituitary D2 mRNA suppression by LG 268, but D2 activity remained intact. In summary, RXR-selective retinoids (rexinoids) have multiple effects on the hypothalamic-pituitary-thyroid axis. Rexinoids directly suppress TSH secretion, TSHbeta mRNA levels and promoter activity, and D2 mRNA levels but have no direct effect on hypothalamic TRH levels. Rexinoids also stimulate type 1 iodothyronine deiodinase activity in the liver and pituitary.


Subject(s)
Hypothalamus/metabolism , Pituitary Gland/metabolism , Retinoid X Receptors/metabolism , Thyroid Gland/metabolism , Animals , Benzoates/pharmacology , Blotting, Western , Cell Line , Chromatin Immunoprecipitation , Dose-Response Relationship, Drug , Iodide Peroxidase/metabolism , Liver/metabolism , Male , Mice , Mice, Transgenic , Organic Chemicals/pharmacology , Promoter Regions, Genetic , RNA, Messenger/metabolism , Retinoids/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Thyrotropin/metabolism , Thyroxine/metabolism , Time Factors , Transfection
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