Early induction of hepatic deiodinase type 1 inhibits hepatosteatosis during NAFLD progression.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) comprises a spectrum ranging from hepatosteatosis to progressive nonalcoholic steatohepatitis that can lead to cirrhosis. Humans with low levels of prohormone thyroxine (T4) have a higher incidence of NAFLD, and thyroid hormone treatment is very promising in all patients with NAFLD. Deiodinase type 1 (Dio1) is a hepatic enzyme that converts T4 to the bioactive T3 and therefore regulates thyroid hormone availability within hepatocytes. We investigated the role of this intrahepatic regulation during the progression of NAFLD. METHODS: We investigated hepatic thyroid hormone metabolism in two NAFLD models: wild-type mice fed a Western diet with fructose and Leprdb mice fed a methionine- and choline-deficient diet. AAV8-mediated liver-specific Dio1 knockdown was employed to investigate the role of Dio1 during the progression of NAFLD. Intrahepatic thyroid hormone levels, deiodinase activity, and metabolic parameters were measured. RESULTS: Dio1 expression and activity were increased in the early stages of NAFLD and were associated with an increased T3/T4 ratio. Prevention of this increase by AAV8-mediated liver-specific Dio1 knockdown increased hepatic triglycerides and cholesterol and decreased the pACC/ACC ratio and acylcarnitine levels, suggesting there was lower ß-oxidation. Dio1 siRNA KD in hepatic cells treated with fatty acids showed increased lipid accumulation and decreased oxidative phosphorylation. CONCLUSION: Hepatic Dio1 gene expression was modulated by dietary conditions, was increased during hepatosteatosis and early NASH, and regulated hepatic triglyceride content. These early adaptations likely represent compensatory mechanisms that reduce hepatosteatosis and prevent NASH progression.

Loss of Selenoprotein Iodothyronine Deiodinase 3 Expression Correlates with Progression of Complete Hydatidiform Mole to Gestational Trophoblastic Neoplasia.

To investigate if differences in imprinting at tropho-microRNA (miRNA) genomic clusters can distinguish between pre-gestational trophoblastic neoplasia cases (pre-GTN) and benign complete hydatidiform mole (CHM) cases at the time of initial uterine evacuation. miRNA sequencing was performed on frozen tissue from 39 CHM cases including 9 GTN cases. DIO3, DLK1, RTL1, and MEG 3 mRNA levels were assessed by qRT-PCR. Protein abundance was assessed by Western blot for DIO3, DLK1, and RTL1. qRT-PCR and Western blot were performed for selenoproteins and markers of oxidative stress. Immunohistochemistry (IHC) was performed for DIO3 on an independent validation set of clinical samples (n = 42) and compared to normal placenta controls across gestational ages. Relative expression of the 14q32 miRNA cluster was lower in pre-GTN cases. There were no differences in protein abundance of DLK1 or RTL1. Notably, there was lower protein expression of DIO3 in pre-GTN cases (5-fold, p < 0.03). There were no differences in mRNA levels of DIO3, DLK1, RTL1 or MEG 3. mRNA levels were higher in all CHM cases compared to normal placenta. IHC showed syncytiotrophoblast-specific DIO3 immunostaining in benign CHM cases and normal placenta, while pre-GTN cases of CHM lacked DIO3 expression. We describe two new biomarkers of pre-GTN CHM cases: decreased 14q32 miRNA expression and loss of DIO3 expression by IHC. Differences in imprinting between benign CHM and pre-GTN cases may provide insight into the fundamental development of CHM.

The Intrinsic Activity of Thyroxine Is Critical for Survival and Growth and Regulates Gene Expression in Neonatal Liver.

Background: Thyroxine (T4) is generally considered to be a prohormone that requires conversion to triiodothyronine (T3) to exert biological activity. Although evidence suggests that T4 has intrinsic activity, it is questionable if this activity has any physiological relevance. Methods: To answer this question, triple knockout (KO) mice (Triples) that cannot express the types 1 (D1) and 2 (D2) deiodinase and the Pax8 genes were generated. Thus, they lack a thyroid and cannot convert T4 to T3. Triples were injected on alternate days with either vehicle or physiological doses of T4, T3, or T3+T4 from postnatal days 2-14. They were euthanized at P15, and RNA-seq was employed to profile gene expression in the liver. In another experiment, Pax8KO mice were injected with T3, T4, or T4+T3, and growth rate and survival to P84 were determined. Results: The growth retardation of Triples was not improved by either T3 or T4 alone but was significantly improved by T4+T3. In the liver, T4 significantly regulated the expression of genes that were also regulated by T3, but the proportion of genes that were negatively regulated was higher in mice treated with T4 than in mice treated with T3. Treatment with T4+T3 identified genes that were regulated synergistically by T3 and T4, and genes that were regulated only by T4+T3. Analysis of these genes revealed enrichment in mechanisms related to cell proliferation and cholesterol physiology, suggesting a unique contribution of T4 to these biological functions. Pax8KO mice all survived to P84 when injected with T4 or T4+T3. However, survival rate with T3 was only 50% and 10% at 3.5 and 12 weeks of life, respectively. Conclusions: T4 has intrinsic activity in vivo and is critical for survival and growth. At a physiological level, T4 per se can upregulate or downregulate many T3 target genes in the neonatal liver. While most of these genes are also regulated by T3, subsets respond exclusively to T4 or demonstrate enhanced or normalized expression only in the presence of both hormones. These studies demonstrate for the first time a complex dependency on both T4 and T3 for normal mammalian growth and development.

Spermatogonial Type 3 Deiodinase Regulates Thyroid Hormone Target Genes in Developing Testicular Somatic Cells.

Premature overexposure to thyroid hormone causes profound effects on testis growth, spermatogenesis, and male fertility. We used genetic mouse models of type 3 deiodinase (DIO3) deficiency to determine the genetic programs affected by premature thyroid hormone action and to define the role of DIO3 in regulating thyroid hormone economy in testicular cells. Gene expression profiling in the neonatal testis of DIO3-deficient mice identified 5699 differentially expressed genes. Upregulated and downregulated genes were, respectively, involved according to DAVID analysis with cell differentiation and proliferation. They included anti-Müllerian hormone and genes involved in the formation of the blood-testis barrier, which are specific to Sertoli cells (SCs). They also included steroidogenic genes, which are specific to Leydig cells. Comparison with published data sets of genes enriched in SCs and spermatogonia, and responsive to retinoic acid (RA), identified a subset of genes that were regulated similarly by RA and thyroid hormone. This subset of genes showed an expression bias, as they were downregulated when enriched in spermatogonia and upregulated when enriched in SCs. Furthermore, using a genetic approach, we found that DIO3 is not expressed in SCs, but spermatogonia-specific inactivation of DIO3 led to impaired testis growth, reduced SC number, decreased cell proliferation and, especially during neonatal development, altered gene expression specific to somatic cells. These findings indicate that spermatogonial DIO3 protects testicular cells from untimely thyroid hormone signaling and demonstrate a mechanism of cross-talk between somatic and germ cells in the neonatal testis that involves the regulation of thyroid hormone availability and action.

Adult onset of type 3 deiodinase deficiency in mice alters brain gene expression and increases locomotor activity.

Constitutive loss of the type 3 deiodinase (DIO3) causes abnormally increased levels of thyroid hormone action in the developing and adult brain, leading to an array of behavioral abnormalities. To determine to what extent those phenotypes derive from a lack of DIO3 in the adult brain, versus developmental consequences, we created a mouse model of conditional DIO3 inactivation. Mice carrying "floxed" Dio3 alleles and a tamoxifen-inducible cre transgene were injected with tamoxifen at two months of age. Compared to oil-injected controls, the brain tissue of these mice showed a 75-80% decrease in DIO3 activity and 85-95% Dio3 mRNA was expressed from recombinant alleles. Mice with adult DIO3 deficiency did not show significant differences in growth, serum thyroid hormone parameters or behaviors related to anxiety and depression. However, female mice exhibited elevated locomotor activity and increased marble-burying behavior. They also manifested relatively modest alterations in the expression of T3-dependent genes and genes related to hyperactivity in a sex- and region-specific manner. Upon thyroid hormone treatment, the expression response of T3-regulated genes was generally more pronounced in DIO3-deficient female mice than in female controls, while the opposite effect of altered genotype was noticed in males. The extent of the molecular and behavioral phenotypes of adult-onset DIO3 deficiency suggests that a substantial proportion of the neurological abnormalities caused by constitutive DIO3 deficiency has a developmental origin. However, our results show that DIO3 in the adult brain also influences behavior and sensitivity to thyroid hormone action in a sexually dimorphic fashion.

Age-Dependent Changes in Glucose Homeostasis in Male Deiodinase Type 2 Knockout Zebrafish.

Thyroid hormones (THs) are crucial regulators of glucose metabolism and insulin sensitivity. Moreover, inactivating mutations in type 2 deiodinase (DIO2), the major TH-activating enzyme, have been associated with type 2 diabetes mellitus in both humans and mice. We studied the link between Dio2 deficiency and glucose homeostasis in fasted males of two different Dio2 knockout (KO) zebrafish lines. Young adult Dio2KO zebrafish (6 to 9 months) were hyperglycemic. Both insulin and glucagon expression were increased, whereas ß and α cell numbers in the main pancreatic islet were similar to those in wild-types. Insulin receptor expression in skeletal muscle was decreased at 6 months, accompanied by a strong downregulation of hexokinase and pyruvate kinase expression. Blood glucose levels in Dio2KO zebrafish, however, normalized around 1 year of age. Older mutants (18 to 24 months) were normoglycemic, and increased insulin and glucagon expression was accompanied by a prominent increase in pancreatic islet size and ß and α cell numbers. Older Dio2KO zebrafish also showed strongly decreased expression of glucagon receptors in the gastrointestinal system as well as decreased expression of glucose transporters GLUT2 and GLUT12, glucose-6-phosphatase, and glycogen synthase 2. This study shows that Dio2KO zebrafish suffer from transient hyperglycemia, which is counteracted with increasing age by a prominent hyperplasia of the endocrine pancreas together with decreases in hepatic glucagon sensitivity and intestinal glucose uptake. Further research on the mechanisms allowing compensation in older Dio2KO zebrafish may help to identify new therapeutic targets for (TH deficiency-related) hyperglycemia.

Adult Mice Lacking Mct8 and Dio2 Proteins Present Alterations in Peripheral Thyroid Hormone Levels and Severe Brain and Motor Skill Impairments.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) lead to peripheral hyperthyroidism and profound psychomotor alterations in humans. Mice lacking Mct8 present peripheral hyperthyroidism but no gross neurological abnormalities due to brain compensatory mechanisms involving the enzyme deiodinase type 2 (Dio2). Methods: Here we have analyzed the endocrine and neurologic phenotype of mice lacking both Mct8 and Dio2 at three and six months of age. Thyroxine (T4) and 3,5,3' triiodothyronine (T3) levels/content were measured by specific radioimmunoassays; motor skill performance was evaluated by the footprint, rotarod, four limb hanging wire, and balance beam tests; and brain histological analysis was performed by immunostaining for neurofilament and parvalbumin. Results: We have found that this mouse model presents peripheral hyperthyroidism and brain hypothyroidism. Interestingly, the severity of the brain hypothyroidism seems permanent and varies across regions, with the striatum being a particularly affected area. We have also found brain alterations at the histological level compatible with TH deficiency and impaired motor skills. Conclusions: These findings indicate the potential of Mct8/Dio2-deficient mice to represent a model for human MCT8 deficiency, to understand the mechanisms underlying its pathophysiology, and ultimately design therapeutic interventions for human patients.

Local hyperthyroidism promotes pancreatic acinar cell proliferation during acute pancreatitis.

Proliferation of pancreatic acinar cells is a critical process in the pathophysiology of pancreatic diseases, because limited or defective proliferation is associated with organ dysfunction and patient morbidity. In this context, elucidating the signalling pathways that trigger and sustain acinar proliferation is pivotal to develop therapeutic interventions promoting the regenerative process of the organ. In this study we used genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones to elucidate their role in acinar proliferation following caerulein-mediated acute pancreatitis in mice. In addition, molecular mechanisms mediating the effects of thyroid hormones were identified by genetic and pharmacological inactivation of selected signalling pathways.In this study we demonstrated that levels of the thyroid hormone 3,3',5-triiodo-l-thyronine (T3) transiently increased in the pancreas during acute pancreatitis. Moreover, by using genetic and pharmacological approaches to manipulate both local and systemic levels of thyroid hormones, we showed that T3 was required to promote proliferation of pancreatic acinar cells, without affecting the extent of tissue damage or inflammatory infiltration.Finally, upon genetic and pharmacological inactivation of selected signalling pathways, we demonstrated that T3 exerted its mitogenic effect on acinar cells via a tightly controlled action on different molecular effectors, including histone deacetylase, AKT, and TGFß signalling.In conclusion, our data suggest that local availability of T3 in the pancreas is required to promote acinar cell proliferation and provide the rationale to exploit thyroid hormone signalling to enhance pancreatic regeneration. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased aggression and lack of maternal behavior in Dio3-deficient mice are associated with abnormalities in oxytocin and vasopressin systems.

Thyroid hormones regulate many aspects of brain development and function, and alterations in the levels of thyroid hormone action lead to abnormal anxiety- and depression-like behaviors. A complement of factors in the brain function independently of circulating levels of hormone to strictly controlled local thyroid hormone signaling. A critical factor is the type 3 deiodinase (DIO3), which is located in neurons and protects the brain from excessive thyroid hormone. Here, we examined whether a local increase in brain thyroid hormone action secondary to DIO3 deficiency is of consequence for social behaviors. Although we did not observe alterations in sociability, Dio3-/- mice of both sexes exhibited a significant increase in aggression-related behaviors and mild deficits in olfactory function. In addition, 85% of Dio3-/- dams manifested no pup-retrieval behavior and increased aggression toward the newborns. The abnormal social behaviors of Dio3-/- mice were associated with sexually dimorphic alterations in the physiology of oxytocin (OXT) and arginine vasopressin (AVP), 2 neuropeptides with important roles in determining social interactions. These alterations included low adult serum levels of OXT and AVP, and an abnormal expression of Oxt, Avp and their receptors in the neonatal and adult hypothalamus. Our results demonstrate that DIO3 is essential for normal aggression and maternal behaviors, and indicate that abnormal local regulation of thyroid hormone action in the brain may contribute to the social deficits associated with neurodevelopmental disorders.

Thyroid Hormone Economy in the Perinatal Mouse Brain: Implications for Cerebral Cortex Development.

Thyroid hormones (THs, T4 and the transcriptionally active hormone T3) play an essential role in neurodevelopment; however, the mechanisms underlying T3 brain delivery during mice fetal development are not well known. This work has explored the sources of brain T3 during mice fetal development using biochemical, anatomical, and molecular approaches. The findings revealed that during late gestation, a large amount of fetal brain T4 is of maternal origin. Also, in the developing mouse brain, fetal T3 content is regulated through the conversion of T4 into T3 by type-2 deiodinase (D2) activity, which is present from earlier prenatal stages. Additionally, D2 activity was found to be essential to mediate expression of T3-dependent genes in the cerebral cortex, and also necessary to generate the transient cerebral cortex hyperthyroidism present in mice lacking the TH transporter Monocarboxylate transporter 8. Notably, the gene encoding for D2 (Dio2) was mainly expressed at the blood-cerebrospinal fluid barrier (BCSFB). Overall, these data signify that T4 deiodinated by D2 may be the only source of T3 during neocortical development. We therefore propose that D2 activity at the BCSFB converts the T4 transported across the choroid plexus into T3, thus supplying the brain with active hormone to maintain TH homeostasis.

Mild thyroid peroxidase deficiency caused by TPO mutations with residual activity: Correlation between clinical phenotypes and enzymatic activity.

Thyroid peroxidase (TPO) deficiency, caused by biallelic TPO mutations, is a well-established genetic form of congenital hypothyroidism (CH). More than 100 patients have been published, and the patients have been diagnosed mostly in the frame of newborn screening (NBS) programs. Correlation between clinical phenotypes and TPO activity remains unclear. Here, we report clinical and molecular findings of two unrelated TPO mutation-carrying mildly hypothyroid patients. The two patients were born at term after an uneventful pregnancy and delivery, and were NBS negative. They sought medical attention due to goiter at age 8 years. Evaluation of the thyroid showed mild elevation of serum TSH levels, normal or slightly low serum T4 levels, high serum T3 to T4 molar ratio, high serum thyroglobulin levels, and high thyroidal 123I uptake. We performed next-generation sequencing-based genetic screening, and found that one patient was compound heterozygous for two novel TPO mutations (p.Asp224del; c.820-2A>G), and the other was homozygous for a previously known mutation (p.Trp527Cys). In vitro functional analyses using HEK293 cells showed that the two amino acid-altering mutations (p.Asp224del and p.Trp527Cys) caused partial loss of the enzymatic activity. In conclusion, we report that TPO mutations with residual activity are associated with mild TPO deficiency, which is clinically characterized by marked goiter, mild TSH elevation, high serum T3 to T4 molar ratio, and high serum thyroglobulin levels. Our findings illuminate the hitherto under-recognized correlation between clinical phenotypes and residual enzymatic activity among patients with TPO deficiency.

Increased anxiety and fear memory in adult mice lacking type 2 deiodinase.

A euthyroid state in the brain is crucial for its adequate development and function. Impairments in thyroid hormones (THs; T3 or 3,5,3'-triiodothyronine and T4 or thyroxine) levels and availability in brain can lead to neurological alterations and to psychiatric disorders, particularly mood disorders. The thyroid gland synthetizes mainly T4, which is secreted to circulating blood, however, most actions of THs are mediated by T3, the transcriptionally active form. In the brain, intracellular concentrations of T3 are modulated by the activity of type 2 (D2) and type 3 (D3) deiodinases. In the present work, we evaluated learning and memory capabilities and anxiety-like behavior at adult stages in mice lacking D2 (D2KO) and we analyzed the impact of D2-deficiency on TH content and on the expression of T3-dependent genes in the amygdala and the hippocampus. We found that D2KO mice do not present impairments in spatial learning and memory, but they display emotional alterations with increased anxiety-like behavior as well as enhanced auditory-cued fear memory and spontaneous recovery of fear memory following extinction. D2KO mice also presented reduced T3 content in the hippocampus and decreased expression of the T3-dependent gene Dio3 in the amygdala suggesting a hypothyroid status in this structure. We propose that the emotional dysfunctions found in D2KO mice can arise from the reduced T3 content in their brain, which consequently leads to alterations in gene expression with functional consequences. We found a downregulation in the gene encoding for the calcium-binding protein calretinin (Calb2) in the amygdala of D2KO mice that could affect the GABAergic transmission. The current findings in D2KO mice can provide insight into emotional disorders present in humans with DIO2 polymorphisms.

Decreased anxiety- and depression-like behaviors and hyperactivity in a type 3 deiodinase-deficient mouse showing brain thyrotoxicosis and peripheral hypothyroidism.

Hypo- and hyperthyroid states, as well as functional abnormalities in the hypothalamic-pituitary-thyroid axis have been associated with psychiatric conditions like anxiety and depression. However, the nature of this relationship is poorly understood since it is difficult to ascertain the thyroid status of the brain in humans. Data from animal models indicate that the brain exhibits efficient homeostatic mechanisms that maintain local levels of the active thyroid hormone, triiodothyronine (T3) within a narrow range. To better understand the consequences of peripheral and central thyroid status for mood-related behaviors, we used a mouse model of type 3 deiodinase (DIO3) deficiency (Dio3 -/- mouse). This enzyme inactivates thyroid hormone and is highly expressed in the adult central nervous system. Adult Dio3 -/- mice exhibit elevated levels of T3-dependent gene expression in the brain, despite peripheral hypothyroidism as indicated by low circulating levels of thyroxine and T3. Dio3 -/- mice of both sexes exhibit hyperactivity and significantly decreased anxiety-like behavior, as measured by longer time spent in the open arms of the elevated plus maze and in the light area of the light/dark box. During the tail suspension, they stayed immobile for a significantly shorter time than their wild-type littermates, suggesting decreased depression-like behavior. These results indicate that increased thyroid hormone in the brain, not necessarily in peripheral tissues, correlates with hyperactivity and with decreases in anxiety and depression-like behaviors. Our results also underscore the importance of DIO3 as a determinant of behavior by locally regulating the brain levels of thyroid hormone.

Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility.

Iodothyronine deiodinases are selenocysteine-containing enzymes that activate or inactivate thyroid hormones (THs). Deiodinase type 2 (Dio2) catalyzes the conversion of the prohormone T4 into the transcriptionally active T3 and is the predominant activating deiodinase in zebrafish. Using zinc finger nucleases, we generated two different dio2(-/-) mutant zebrafish lines to investigate the physiological function of this TH activator. The first line contains a deletion of 9 bp, resulting in an in-frame elimination of three conserved amino acids. The other line is characterized by an insertion of 4 bp, leading to the introduction of a premature stop-codon. Both lines completely lack Dio2 activity, resulting in a strong reduction of T3 abundancy in all tissues tested. Early development is clearly perturbed in these animals, as shown by a diverse set of morphometric parameters, defects in swim bladder inflation, and disturbed locomotor activity tested between 1 and 7 days after fertilization. Permanent Dio2 deficiency also provokes long-term effects because growth and especially fertility are severely hampered. Possible compensatory mechanisms were investigated in adult dio2(-/-) mutants, revealing a down-regulation of the inactivating deiodinase Dio3 and TH receptor transcript levels. As the first nonmammalian model with permanent Dio2 deficiency, these mutant zebrafish lines provide evidence that Dio2 is essential to assure normal development and to obtain a normal adult phenotype.

Type 2 Deiodinase Disruption in Astrocytes Results in Anxiety-Depressive-Like Behavior in Male Mice.

Millions of levothyroxine-treated hypothyroid patients complain of impaired cognition despite normal TSH serum levels. This could reflect abnormalities in the type 2 deiodinase (D2)-mediated T4-to-T3 conversion, given their much greater dependence on the D2 pathway for T3 production. T3 normally reaches the brain directly from the circulation or is produced locally by D2 in astrocytes. Here we report that mice with astrocyte-specific Dio2 inactivation (Astro-D2KO) have normal serum T3 but exhibit anxiety-depression-like behavior as found in open field and elevated plus maze studies and when tested for depression using the tail-suspension and the forced-swimming tests. Remarkably, 4 weeks of daily treadmill exercise sessions eliminated this phenotype. Microarray gene expression profiling of the Astro-D2KO hippocampi identified an enrichment of three gene sets related to inflammation and impoverishment of three gene sets related to mitochondrial function and response to oxidative stress. Despite normal neurogenesis, the Astro-D2KO hippocampi exhibited decreased expression of four of six known to be positively regulated genes by T3, ie, Mbp (∼43%), Mag (∼34%), Hr (∼49%), and Aldh1a1 (∼61%) and increased expression of 3 of 12 genes negatively regulated by T3, ie, Dgkg (∼17%), Syce2 (∼26%), and Col6a1 (∼3-fold) by quantitative real-time PCR. Notably, in Astro-D2KO animals, there was also a reduction in mRNA levels of genes known to be affected in classical animal models of depression, ie, Bdnf (∼18%), Ntf3 (∼43%), Nmdar (∼26%), and GR (∼20%), which were also normalized by daily exercise sessions. These findings suggest that defects in Dio2 expression in the brain could result in mood and behavioral disorders.

Aberrant Calreticulin Expression in Articular Cartilage of Dio2 Deficient Mice.

OBJECTIVE: To identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis. METHODS: Previously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition. RESULTS: Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > |1,5|) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition. CONCLUSION: We here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

Reciprocal interplay between thyroid hormone and microRNA-21 regulates hedgehog pathway-driven skin tumorigenesis.

The thyroid hormone-inactivating (TH-inactivating) enzyme type 3 iodothyronine deiodinase (D3) is an oncofetal protein that is rarely expressed in adult life but has been shown to be reactivated in the context of proliferation and neoplasms. D3 terminates TH action within the tumor microenvironment, thereby enhancing cancer cell proliferation. However, the pathological role of D3 and the contribution of TH metabolism in cancer have yet to be fully explored. Here, we describe a reciprocal regulation between TH action and the cancer-associated microRNA-21 (miR21) in basal cell carcinoma (BCC) skin tumors. We found that, besides being negatively regulated by TH at the transcriptional level, miR21 attenuates the TH signal by increasing D3 levels. The ability of miR21 to positively regulate D3 was mediated by the tumor suppressor gene GRHL3, a hitherto unrecognized D3 transcriptional inhibitor. Finally, in a BCC mouse model, keratinocyte-specific D3 depletion markedly reduced tumor growth. Together, our results establish TH action as a critical hub of multiple oncogenic pathways and provide functional and mechanistic evidence of the involvement of TH metabolism in BCC tumorigenesis. Moreover, our results identify a miR21/GRHL3/D3 axis that reduces TH in the tumor microenvironment and has potential to be targeted as a therapeutic approach to BCC.

Type 1 Deiodinase Regulates ApoA-I Gene Expression and ApoA-I Synthesis Independent of Thyroid Hormone Signaling.

OBJECTIVE: Plasma levels of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) are reduced in individuals with defective insulin signaling. Initial studies using liver-specific insulin receptor (InsR) knockout mice identified reduced expression of type 1 deiodinase (Dio1) as a potentially novel link between defective hepatic insulin signaling and reduced expression of the ApoA-I gene. Our objective was to examine the regulation of ApoA-I expression by Dio1. APPROACH AND RESULTS: Acute inactivation of InsR by adenoviral delivery of Cre recombinase to InsR floxed mice reduced HDL-C and expression of both ApoA-I and Dio1. Overexpression of Dio1 in InsR knockout mice restored HDL-C and ApoA-I levels and increased the expression of ApoA-I. Dio1 knockout mice had low expression of ApoA-I and reduced serum levels of HDL-C and ApoA-I. Treatment of C57BL/6J mice with antisense to Dio1 reduced ApoA-I mRNA, HDL-C, and serum ApoA-I. Hepatic 3,5,3'-triiodothyronine content was normal or elevated in InsR knockout mice or Dio1 knockout mice. Knockdown of either InsR or Dio1 by siRNA in HepG2 cells decreased the expression of ApoA-I and ApoA-I synthesis and secretion. siRNA knockdown of InsR or Dio1 decreased activity of a region of the ApoA-I promoter lacking thyroid hormone response elements (region B). Electrophoretic mobility shift assay demonstrated that reduced Dio1 expression decreased the binding of nuclear proteins to region B. CONCLUSIONS: Reductions in Dio1 expression reduce the expression of ApoA-I in a 3,5,3'-triiodothyronine-/thyroid hormone response element-independent manner.