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. High mobility group (HMG)1 proteins constitute a significant proportion of non-histone proteins of eukaryotic chromatin. They are abundant proteins that are grouped, in part based on their DNA binding characteristics, into three major classes, HMGA, HMGB, and HMGN (1-4). HMGB proteins contain one or more homologous repeats of the ϳ80-amino acid sequence HMG box and are classified into two families based on the abundance, function, and DNA specificity of this conserved region (1, 5, 6). The moderately sequence-specific family is typified by transcription factors such as sex-determining factor SRY and lymphoid enhancer factor LEF-1 (7, 8), whereas the non-sequence-specific family is represented by so-called architectural factors HMGB1/2 and the Saccharomyces cerevisiae non-histone chromosomal proteins 6A and 6B (NHP6A/B) (9).The tertiary structures of HMG boxes from sequence-specific and non-sequence-specific proteins have revealed an evolutionarily conserved, common global fold consisting of an L-shaped structure composed of three ␣-helices (10 -19). The HMG DNA binding domain, which interacts with ϳ10 bp of duplex, binds to the minor groove of DNA by partial intercalation of one or two surface-exposed, conserved hydrophobic residues into the base pair stack. Consequently, the DNA is greatly distorted, resulting in a sharp bend and helical underwinding (4,8,14,16,20). SRY and LEF-1 cause bending by insertion of helix I hydrophobic residues Ile and Met, respectively, into the base pair stack (Fig. 1) (8, 17). HMGB1 contains tandem HMG box domains referred to as box A and box B; DNA-intercalating residues are located...

Summary 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.

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.

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.

BackgroundNuclear factor-κB (NF-κB) is constitutively activated in many cancers and plays a key role in promoting cell proliferation, survival, and invasion. Our understanding of NF-κB signaling in thyroid cancer, however, is limited. In this study, we have investigated the role of NF-κB signaling in thyroid cancer cell proliferation, invasion, and apoptosis using selective genetic inhibition of NF-κB in advanced thyroid cancer cell lines.ResultsThree pharmacologic inhibitors of NF-κB differentially inhibited growth in a panel of advanced thyroid cancer cell lines, suggesting that these NF-κB inhibitors may have off-target effects. We therefore used a selective genetic approach to inhibit NF-κB signaling by overexpression of a dominant-negative IκBα (mIκBα). 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 TNFα-induced apoptosis was observed in all cell lines, likely through an NF-κB-dependent mechanism. Inhibition of NF-κB by mIκBα sensitized a subset of cell lines to TNFα-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-κB inhibition by mIκBα 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 mIκBα expression in all cell lines tested.ConclusionsThese data indicate that selective inhibition of NF-κB 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-κB signaling alone. Instead, our findings suggest that other important molecular processes play a critical role in defining the extent of NF-κB function within cancer cells.

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 mm3 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.

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.

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.