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Repair-related activation of hedgehog signaling in stromal cells promotes intrahepatic hypothyroidism.

Bohinc BN, Michelotti G, Xie G, Pang H, Suzuki A, Guy CD, Piercy D, Kruger L, Swiderska-Syn M, Machado M, Pereira T, Zavacki AM, Abdelmalek M, Diehl AM - Endocrinology (2014)

Bottom Line: Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression.Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4.In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Endocrinology, Diabetes, and Metabolism (B.N.B., D.P.) and Gastroenterology (G.M., G.X., A.S., L.K., M.S.-S., M.M., T.P., M.A., A.M.D.) and Departments of Biostatistics and Bioinformatics (H.P.) and Pathology (C.D.G.), Duke University, Durham, North Carolina 27710; and Division of Endocrinology, Diabetes, and Metabolism (A.M.Z.), Brigham and Women's Hospital, Boston, Massachusetts 02115.

ABSTRACT
Thyroid hormone (TH) is important for tissue repair because it regulates cellular differentiation. Intrahepatic TH activity is controlled by both serum TH levels and hepatic deiodinases. TH substrate (T4) is converted into active hormone (T3) by deiodinase 1 (D1) but into inactive hormone (rT3) by deiodinase 3 (D3). Although the relative expressions of D1 and D3 are known to change during liver injury, the cell types and signaling mechanisms involved are unclear. We evaluated the hypothesis that changes in hepatic deiodinases result from repair-related activation of the Hedgehog pathway in stromal cells. We localized deiodinase expression, assessed changes during injury, and determined how targeted manipulation of Hedgehog signaling in stromal cells impacted hepatic deiodinase expression, TH content, and TH action in rodents. Humans with chronic liver disease were also studied. In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, whereas stromal expression of D3 increased, particularly in myofibroblasts. Conditionally disrupting Hedgehog signaling in myofibroblasts normalized deiodinase expression. Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression. In patients, this was reflected by increased serum rT3. Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4. In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

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Myofibroblastic hepatic stellate cells are major sources of D3. A, Costaining for D3 (brown) with desmin (green) in representative sham-operated rats and rats at different time points after BDL. Double-positive cells were counted, expressed as a percentage of cells per high-power field (HPF), and graphed as mean ± SEM. **, P < .01. B, qRT-PCR analysis of DIO1 and DIO3 mRNA at different times during culture of primary rat stellate cells. *, P < .05; **, P < .01 vs day 0. C, Fluorescence-activated cell sorting analysis of primary stellate cells is shown. Numbers of D1- or D3-positive cells are demonstrated relative to respective IgG-stained controls.
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Figure 4: Myofibroblastic hepatic stellate cells are major sources of D3. A, Costaining for D3 (brown) with desmin (green) in representative sham-operated rats and rats at different time points after BDL. Double-positive cells were counted, expressed as a percentage of cells per high-power field (HPF), and graphed as mean ± SEM. **, P < .01. B, qRT-PCR analysis of DIO1 and DIO3 mRNA at different times during culture of primary rat stellate cells. *, P < .05; **, P < .01 vs day 0. C, Fluorescence-activated cell sorting analysis of primary stellate cells is shown. Numbers of D1- or D3-positive cells are demonstrated relative to respective IgG-stained controls.

Mentions: Among liver stromal cells, injury-activated HSCs play particularly pivotal roles in hepatic wound healing/regeneration. In addition to becoming a major source of fibrogenic myofibroblasts, activated HSCs produce various hepatocyte growth factors, chemokines, and mediators of angiogenesis, and there is strong experimental evidence that inhibiting HSC activation drastically impairs liver regeneration (20, 21). Double immunostaining for desmin, a stellate cell-specific marker (22), and D3 confirmed that many of the D3-expressing cells that accumulated after BDL were HSCs (Figure 4A), raising the possibility that HSC might up-regulate their expression of D3 as they activate during injury. To evaluate this more directly, we compared D3 expression in primary HSC at different time points during culture-induced activation. Both DIO3 mRNA (Figure 4B) and D3 protein (Figure 4C) levels increased as quiescent HSCs became myofibroblasts. This activation-related induction of D3 expression was accompanied by a reduced expression of DIO1 mRNA and D1 protein (Figure 4, B and C). Thus, HSC activation is characterized by reciprocal changes in the expression of DIO1 and DIO3, with the resultant predominance in DIO3 predicted to reduce accumulation of biologically-active TH.


Repair-related activation of hedgehog signaling in stromal cells promotes intrahepatic hypothyroidism.

Bohinc BN, Michelotti G, Xie G, Pang H, Suzuki A, Guy CD, Piercy D, Kruger L, Swiderska-Syn M, Machado M, Pereira T, Zavacki AM, Abdelmalek M, Diehl AM - Endocrinology (2014)

Myofibroblastic hepatic stellate cells are major sources of D3. A, Costaining for D3 (brown) with desmin (green) in representative sham-operated rats and rats at different time points after BDL. Double-positive cells were counted, expressed as a percentage of cells per high-power field (HPF), and graphed as mean ± SEM. **, P < .01. B, qRT-PCR analysis of DIO1 and DIO3 mRNA at different times during culture of primary rat stellate cells. *, P < .05; **, P < .01 vs day 0. C, Fluorescence-activated cell sorting analysis of primary stellate cells is shown. Numbers of D1- or D3-positive cells are demonstrated relative to respective IgG-stained controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256825&req=5

Figure 4: Myofibroblastic hepatic stellate cells are major sources of D3. A, Costaining for D3 (brown) with desmin (green) in representative sham-operated rats and rats at different time points after BDL. Double-positive cells were counted, expressed as a percentage of cells per high-power field (HPF), and graphed as mean ± SEM. **, P < .01. B, qRT-PCR analysis of DIO1 and DIO3 mRNA at different times during culture of primary rat stellate cells. *, P < .05; **, P < .01 vs day 0. C, Fluorescence-activated cell sorting analysis of primary stellate cells is shown. Numbers of D1- or D3-positive cells are demonstrated relative to respective IgG-stained controls.
Mentions: Among liver stromal cells, injury-activated HSCs play particularly pivotal roles in hepatic wound healing/regeneration. In addition to becoming a major source of fibrogenic myofibroblasts, activated HSCs produce various hepatocyte growth factors, chemokines, and mediators of angiogenesis, and there is strong experimental evidence that inhibiting HSC activation drastically impairs liver regeneration (20, 21). Double immunostaining for desmin, a stellate cell-specific marker (22), and D3 confirmed that many of the D3-expressing cells that accumulated after BDL were HSCs (Figure 4A), raising the possibility that HSC might up-regulate their expression of D3 as they activate during injury. To evaluate this more directly, we compared D3 expression in primary HSC at different time points during culture-induced activation. Both DIO3 mRNA (Figure 4B) and D3 protein (Figure 4C) levels increased as quiescent HSCs became myofibroblasts. This activation-related induction of D3 expression was accompanied by a reduced expression of DIO1 mRNA and D1 protein (Figure 4, B and C). Thus, HSC activation is characterized by reciprocal changes in the expression of DIO1 and DIO3, with the resultant predominance in DIO3 predicted to reduce accumulation of biologically-active TH.

Bottom Line: Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression.Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4.In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

View Article: PubMed Central - PubMed

Affiliation: Divisions of Endocrinology, Diabetes, and Metabolism (B.N.B., D.P.) and Gastroenterology (G.M., G.X., A.S., L.K., M.S.-S., M.M., T.P., M.A., A.M.D.) and Departments of Biostatistics and Bioinformatics (H.P.) and Pathology (C.D.G.), Duke University, Durham, North Carolina 27710; and Division of Endocrinology, Diabetes, and Metabolism (A.M.Z.), Brigham and Women's Hospital, Boston, Massachusetts 02115.

ABSTRACT
Thyroid hormone (TH) is important for tissue repair because it regulates cellular differentiation. Intrahepatic TH activity is controlled by both serum TH levels and hepatic deiodinases. TH substrate (T4) is converted into active hormone (T3) by deiodinase 1 (D1) but into inactive hormone (rT3) by deiodinase 3 (D3). Although the relative expressions of D1 and D3 are known to change during liver injury, the cell types and signaling mechanisms involved are unclear. We evaluated the hypothesis that changes in hepatic deiodinases result from repair-related activation of the Hedgehog pathway in stromal cells. We localized deiodinase expression, assessed changes during injury, and determined how targeted manipulation of Hedgehog signaling in stromal cells impacted hepatic deiodinase expression, TH content, and TH action in rodents. Humans with chronic liver disease were also studied. In healthy liver, hepatocytes strongly expressed D1 and stromal cells weakly expressed D3. During injury, hepatocyte expression of D1 decreased, whereas stromal expression of D3 increased, particularly in myofibroblasts. Conditionally disrupting Hedgehog signaling in myofibroblasts normalized deiodinase expression. Repair-related changes in deiodinases were accompanied by reduced hepatic TH content and TH-regulated gene expression. In patients, this was reflected by increased serum rT3. Moreover, the decreases in the free T3 to rT3 and free T4 to rT3 ratios distinguished advanced from mild fibrosis, even in individuals with similar serum levels of TSH and free T4. In conclusion, the Hedgehog-dependent changes in liver stromal cells drive repair-related changes in hepatic deiodinase expression that promote intrahepatic hypothyroidism, thereby limiting exposure to T3, an important factor for cellular differentiation.

Show MeSH
Related in: MedlinePlus