Embryonic vitamin D deficiency programs hematopoietic stem cells to induce type 2 diabetes.

Environmental factors may alter the fetal genome to cause metabolic diseases. It is unknown whether embryonic immune cell programming impacts the risk of type 2 diabetes in later life. We demonstrate that transplantation of fetal hematopoietic stem cells (HSCs) made vitamin D deficient in utero induce diabetes in vitamin D-sufficient mice. Vitamin D deficiency epigenetically suppresses Jarid2 expression and activates the Mef2/PGC1a pathway in HSCs, which persists in recipient bone marrow, resulting in adipose macrophage infiltration. These macrophages secrete miR106-5p, which promotes adipose insulin resistance by repressing PIK3 catalytic and regulatory subunits and down-regulating AKT signaling. Vitamin D-deficient monocytes from human cord blood have comparable Jarid2/Mef2/PGC1a expression changes and secrete miR-106b-5p, causing adipocyte insulin resistance. These findings suggest that vitamin D deficiency during development has epigenetic consequences impacting the systemic metabolic milieu.

Non-classical Vitamin D Actions for Renal Protection.

Chronic Kidney Disease (CKD), a disorder that affects 11% of the world's population, is characterized by an acceleration in skeletal, immune, renal, and cardiovascular aging that increases the risk of cardiovascular mortality by 10- to 20-fold, compared to that in individuals with normal renal function. For more than two decades, the progressive impairment in renal capacity to maintain normal circulating levels of the hormonal form of vitamin D (1,25-dihydroxyvitamin D or calcitriol) was considered the main contributor to the reduced survival of CKD patients. Accordingly, calcitriol administration was the treatment of choice to attenuate the progression of secondary hyperparathyroidism (SHPT) and its adverse impact on bone health and vascular calcification. The development of calcitriol analogs, designed to mitigate the resistance to calcitriol suppression of PTH associated with CKD progression, demonstrated survival benefits unrelated to the control of SHPT or skeletal health. The exhaustive search for the pathophysiology behind survival benefits associated with active vitamin D analogs has identified novel anti-inflammatory, anti-hypertensive, anti-aging actions of the vitamin D endocrine system. A major paradigm shift regarding the use of calcitriol or active vitamin D analogs to improve survival in CKD patients emerged upon demonstration of a high prevalence of vitamin D (not calcitriol) deficiency at all stages of CKD and, more significantly, that maintaining serum levels of the calcitriol precursor, 25(OH)vitamin D, above 23 ng/ml delayed CKD progression. The cause of vitamin D deficiency in CKD, however, is unclear since vitamin D bioactivation to 25(OH)D occurs mostly at the liver. Importantly, neither calcitriol nor its analogs can correct vitamin D deficiency. The goals of this chapter are to present our current understanding of the pathogenesis of vitamin D deficiency in CKD and of the causal link between defective vitamin D bioactivation to calcitriol and the onset of molecular pathways that promote CKD progression independently of the degree of SHPT. An understanding of these mechanisms will highlight the need for identification of novel sensitive biomarkers to assess the efficacy of interventions with vitamin D and/or calcitriol(analogs) to ameliorate CKD progression in a PTH-independent manner.

Role of Mineralocorticoid Receptor in Adipogenesis and Obesity in Male Mice.

Increased visceral adiposity and hyperglycemia, 2 characteristics of metabolic syndrome, are also present in conditions of excess glucocorticoids (GCs). GCs are hormones thought to act primarily via the glucocorticoid receptor (GR). GCs are commonly prescribed for inflammatory disorders, yet their use is limited due to many adverse metabolic side effects. In addition to GR, GCs also bind the mineralocorticoid receptor (MR), but there are many conflicting studies about the exact role of MR in metabolic disease. Using MR knockout mice (MRKO), we find that both white and brown adipose depots form normally when compared with wild-type mice at P5. We created mice with adipocyte-specific deletion of MR (FMRKO) to better understand the role of MR in metabolic dysfunction. Treatment of mice with excess GCs for 4 weeks, via corticosterone in drinking water, induced increased fat mass and glucose intolerance to similar levels in FMRKO and floxed control mice. Separately, when fed a high-fat diet for 16 weeks, FMRKO mice had reduced body weight, fat mass, and hepatic steatosis, relative to floxed control mice. Decreased adiposity likely resulted from increased energy expenditure since food intake was not different. RNA sequencing analysis revealed decreased enrichment of genes associated with adipogenesis in inguinal white adipose of FMRKO mice. Differentiation of mouse embryonic fibroblasts (MEFs) showed modestly impaired adipogenesis in MRKO MEFs compared with wild type, but this was rescued upon the addition of peroxisome proliferator-activated receptor gamma (PPARγ) agonist or PPARγ overexpression. Collectively, these studies provide further evidence supporting the potential value of MR as a therapeutic target for conditions associated with metabolic syndrome.

Glucocorticoid Receptor Signaling Is Not Required for In Vivo Adipogenesis.

Regulation of adipogenesis is of major interest given that adipose tissue expansion and dysfunction are central to metabolic syndrome. Glucocorticoids (GCs) are important for adipogenesis in vitro. However, establishing a role for GCs in adipogenesis in vivo has been difficult. GC receptor (GR)‒null mice die at birth, a time at which wild-type (WT) mice do not have fully developed white adipose depots. We conducted several studies aimed at defining the role of GC signaling in adipogenesis in vitro and in vivo. First, we showed that GR-null mouse embryonic fibroblasts (MEFs) have compromised ability to form adipocytes in vitro, a phenotype that can be partially rescued with a peroxisome proliferator-activated receptor γ agonist. Next, we demonstrated that MEFs are capable of forming de novo fat pads in mice despite the absence of GR or circulating GCs [by bilateral adrenalectomy (ADX)]. However, ADX and GR-null fat pads and their associated adipocyte areas were smaller than those in controls. Second, using adipocyte-specific luciferase reporter mice, we identified adipocytes in both WT and GR-null embryonic day (E)18 mouse embryos. Lastly, positive perilipin staining in WT and GR-null E18 embryos confirmed the presence of early white inguinal and brown adipocytes. Taken together, these results provide compelling evidence that GCs and GR augment but are not required for the development of functional adipose tissue in vivo.

Hypothalamic-Pituitary Axis Regulates Hydrogen Sulfide Production.

Decreased growth hormone (GH) and thyroid hormone (TH) signaling are associated with longevity and metabolic fitness. The mechanisms underlying these benefits are poorly understood, but may overlap with those of dietary restriction (DR), which imparts similar benefits. Recently we discovered that hydrogen sulfide (H2S) is increased upon DR and plays an essential role in mediating DR benefits across evolutionary boundaries. Here we found increased hepatic H2S production in long-lived mouse strains of reduced GH and/or TH action, and in a cell-autonomous manner upon serum withdrawal in vitro. Negative regulation of hepatic H2S production by GH and TH was additive and occurred via distinct mechanisms, namely direct transcriptional repression of the H2S-producing enzyme cystathionine γ-lyase (CGL) by TH, and substrate-level control of H2S production by GH. Mice lacking CGL failed to downregulate systemic T4 metabolism and circulating IGF-1, revealing an essential role for H2S in the regulation of key longevity-associated hormones.

Glucocorticoids and Diabetes.

Glucocorticoids (GCs), stress hormones produced by the adrenal gland, are involved in many pathways in physiology and metabolism including glucose homeostasis and inflammation. Excess GC signaling results in Cushing's syndrome and possibly metabolic syndrome. Diabetes, central adiposity, and hyperlipidemia are components of both syndromes. Here, we discuss the mechanisms of GC action, clinical syndromes of GC excess, modulation of glucose homeostasis by GCs, and future treatments for diabetes based on GC signaling.

Characterization of thyroid cancer cell lines in murine orthotopic and intracardiac metastasis models.

Thyroid cancer incidence has been increasing over time, and it is estimated that ∼1950 advanced thyroid cancer patients will die of their disease in 2015. To combat this disease, an enhanced understanding of thyroid cancer development and progression as well as the development of efficacious, targeted therapies are needed. In vitro and in vivo studies utilizing thyroid cancer cell lines and animal models are critically important to these research efforts. In this report, we detail our studies with a panel of authenticated human anaplastic and papillary thyroid cancer (ATC and PTC) cell lines engineered to express firefly luciferase in two in vivo murine cancer models-an orthotopic thyroid cancer model as well as an intracardiac injection metastasis model. In these models, primary tumor growth in the orthotopic model and the establishment and growth of metastases in the intracardiac injection model are followed in vivo using an IVIS imaging system. In the orthotopic model, the ATC cell lines 8505C and T238 and the PTC cell lines K1/GLAG-66 and BCPAP had take rates >90 % with final tumor volumes ranging 84-214 mm(3) over 4-5 weeks. In the intracardiac model, metastasis establishment was successful in the ATC cell lines HTh74, HTh7, 8505C, THJ-16T, and Cal62 with take rates ≥70 %. Only one of the PTC cell lines tested (BCPAP) was successful in the intracardiac model with a take rate of 30 %. These data will be beneficial to inform the choice of cell line and model system for the design of future thyroid cancer studies.

Nuclear factor κB-dependent regulation of angiogenesis, and metastasis in an in vivo model of thyroid cancer is associated with secreted interleukin-8.

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.

PPARγ Promotes Growth and Invasion of Thyroid Cancer Cells.

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.

Inhibition of nuclear factor-kappa B differentially affects thyroid cancer cell growth, apoptosis, and invasion.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is constitutively activated in many cancers and plays a key role in promoting cell proliferation, survival, and invasion. Our understanding of NF-kappaB signaling in thyroid cancer, however, is limited. In this study, we have investigated the role of NF-kappaB signaling in thyroid cancer cell proliferation, invasion, and apoptosis using selective genetic inhibition of NF-kappaB in advanced thyroid cancer cell lines. RESULTS: Three pharmacologic inhibitors of NF-kappaB differentially inhibited growth in a panel of advanced thyroid cancer cell lines, suggesting that these NF-kappaB inhibitors may have off-target effects. We therefore used a selective genetic approach to inhibit NF-kappaB signaling by overexpression of a dominant-negative IkappaBalpha (mIkappaBalpha). These studies revealed decreased cell growth in only one of five thyroid cancer cell lines (8505C), which occurred through a block in the S-G2/M transition. Resistance to TNFalpha-induced apoptosis was observed in all cell lines, likely through an NF-kappaB-dependent mechanism. Inhibition of NF-kappaB by mIkappaBalpha sensitized a subset of cell lines to TNFalpha-induced apoptosis. Sensitive cell lines displayed sustained activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway, defining a potential mechanism of response. Finally, NF-kappaB inhibition by mIkappaBalpha expression differentially reduced thyroid cancer cell invasion in these thyroid cancer cell lines. Sensitive cell lines demonstrated approximately a two-fold decrease in invasion, which was associated with differential expression of MMP-13. MMP-9 was reduced by mIkappaBalpha expression in all cell lines tested. CONCLUSIONS: These data indicate that selective inhibition of NF-kappaB represents an attractive therapeutic target for the treatment of advanced thyroid. However, it is apparent that global regulation of thyroid cancer cell growth and invasion is not achieved by NF-kappaB signaling alone. Instead, our findings suggest that other important molecular processes play a critical role in defining the extent of NF-kappaB function within cancer cells.

Interactions between N- and C-terminal domains of the Saccharomyces cerevisiae high-mobility group protein HMO1 are required for DNA bending.

The Saccharomyces cerevisiae high-mobility group protein HMO1 is composed of two DNA-binding domains termed box A and box B, of which only box B is predicted to adopt a HMG fold, and a lysine-rich C-terminal extension. To assess the interaction between individual domains and their contribution to DNA binding, several HMO1 variants were analyzed. Using circular dichroism spectroscopy, thermal stability was measured. While the melting temperatures of HMO1-boxA and HMO1-boxB are 57.2 and 47.2 degrees C, respectively, HMO1-boxBC, containing box B and the entire C-terminal tail, melts at 46.1 degrees C, suggesting little interaction between box B and the tail. In contrast, full-length HMO1 exhibits a single melting transition at 47.9 degrees C, indicating that interaction between box A and either box B or the tail destabilizes this domain. As HMO1-boxAB, lacking only the lysine-rich C-terminal segment, exhibits two melting transitions at 46.0 and 63.3 degrees C, we conclude that the destabilization of the box A domain seen in full-length HMO1 is due primarily to its interaction with the lysine-rich tail. Determination of DNA substrate specificity using electrophoretic mobility shift assays shows unexpectedly that the lysine-rich tail does not increase DNA binding affinity but instead is required for DNA bending by full-length HMO1; HMO1-boxBC, lacking the box A domain, also fails to bend DNA. In contrast, both HMO1 and HMO1-boxAB, but not the individual HMG domains, exhibit preferred binding to constrained DNA minicircles. Taken together, our data suggest that interactions between box A and the C-terminal tail induce a conformation that is required for DNA bending.

The Saccharomyces cerevisiae high mobility group box protein HMO1 contains two functional DNA binding domains.

High mobility group box (HMGB) proteins are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as 4-way junctions. HMO1 is one of 10 Saccharomyces cerevisiae HMGB proteins, and it is required for normal growth and plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease. Using electrophoretic mobility shift assays, we have shown here that HMO1 binds 26-bp duplex DNA with K(d) = 39.6 +/- 5.0 nm and that its divergent box A domain participates in DNA interactions, albeit with low affinity. HMO1 has only modest preference for DNA with altered conformations, including DNA with nicks, gaps, overhangs, or loops, as well as for 4-way junction structures and supercoiled DNA. HMO1 binds 4-way junctions with half-maximal saturation of 19.6 +/- 2.2 nm, with only a modest increase in affinity in the absence of magnesium ions (half-maximal saturation 6.1 +/- 1.1 nm). Whereas the box A domain contributes modest structure-specific binding, the box B domain is required for high affinity binding. HMO1 bends DNA, as measured by DNA cyclization assays, facilitating cyclization of 136-, 105-, and 87-bp DNA, but not 75-bp DNA, and it has a significantly longer residence time on DNA minicircles compared with linear duplex DNA. The unique DNA binding properties of HMO1 are consistent with global roles in the maintenance of chromatin structure.