Novel Information on the Epitope of an Inverse Agonist Monoclonal Antibody Provides Insight into the Structure of the TSH Receptor

The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity

Constitutive Activation of the Thyroid-Stimulating Hormone Receptor (TSHR) by Mutating Ile691 in the Cytoplasmic Tail Segment

/inositol phosphate (IP) pathways, which stimulate thyroid hormone production and thyroid proliferation./IP signaling pathway./IP cascade

Novel Pathway of Adipogenesis through Cross-Talk between Adipose Tissue Macrophages, Adipose Stem Cells and Adipocytes: Evidence of Cell Plasticity

INTRODUCTION: Previous studies highlight a complex relationship between lineage and phenotype for adipose tissue macrophages (ATMs), adipose stem cells (ASCs), and adipocytes, suggesting a high degree of plasticity of these cells. In the present study, using a novel co-culture system, we further characterized the interaction between ATMs, ASCs and adipocytes. RESEARCH DESIGN AND METHODS: Human adipocytes and the stromal vascular fraction containing ATMs and ASCs were isolated from human adipose tissue and co-cultured for 24 hours. FACS was used to characterize ATMs and ASCs before and after co-culture. Preadipocytes generated after co-culture were characterized by immunostaining for DLK (preadipocytes), CD14 and CD68 (ATMs), CD34 (ASCs), and Nile Red staining for lipid drops. qRT-PCR was used to quantify adipogenic markers such as C/EBPα and PPARγ. A novel fluorescent nanobead lineage tracing method was utilized before co-culture where fluorescent nanobeads were internalized by CD68 (+) ATMs. RESULTS: Co-culture of adipocytes with ATMs and ASCs increased the formation of new preadipocytes, thereby increasing lipid accumulation and C/EBPα and PPARγ gene expression. Preadipocytes originating after co-culture were positive for markers of preadipocytes, ATMs and ASCs. Moreover, fluorescent nanobeads were internalized by ATMs before co-culture and the new preadipocytes formed after co-culture also contained fluorescent nanobeads, suggesting that new preadipocytes originated in part from ATMs. The formation of CD34(+)/CD68(+)/DLK (+) cell spheres supported the interaction of ATMs, ASCs and preadipocytes. CONCLUSIONS: Cross-talk between adipocytes, ATMs and ASCs promotes preadipocyte formation. The regulation of this novel adipogenic pathway involves differentiation of ATMs to preadipocytes. The presence of CD34(+)/CD68(+)/DLK(+) cells grouped in spheres suggest that paracrine interactions between these cell types plays an important role in the generation and proliferation of new preadipocytes. This phenomenon may reflect the in vivo plasticity of adipose tissue in which ATMs play an additional role during inflammation and other disease states. Understanding this novel pathway could influence adipogenesis, leading to new treatments for obesity, inflammation, and type 2 diabetes

The Role of Paracrine and Autocrine Signaling in the Early Phase of Adipogenic Differentiation of Adipose-derived Stem Cells.

INTRODUCTION: High cell density is known to enhance adipogenic differentiation of mesenchymal stem cells, suggesting secretion of signaling factors or cell-contact-mediated signaling. By employing microfluidic biochip technology, we have been able to separate these two processes and study the secretion pathways. METHODS AND RESULTS: Adipogenic differentiation of human adipose-derived stem cells (ASCs) cultured in a microfluidic system was investigated under perfusion conditions with an adipogenic medium or an adipogenic medium supplemented with supernatant from differentiating ASCs (conditioned medium). Conditioned medium increased adipogenic differentiation compared to adipogenic medium with respect to accumulation of lipid-filled vacuoles and gene expression of key adipogenic markers (C/EBPα, C/EBPβ, C/EBPδ, PPARγ, LPL and adiponectin). The positive effects of conditioned medium were observed early in the differentiation process. CONCLUSIONS: Using different cell densities and microfluidic perfusion cell cultures to suppress the effects of cell-released factors, we have demonstrated the significant role played by auto- or paracrine signaling in adipocyte differentiation. The cell-released factor(s) were shown to act in the recruitment phase of the differentiation process

Polycystic ovary syndrome

The document attached has been archived with permission from the editor of the Medical Journal of Australia. An external link to the publisher’s copy is included.Polycystic ovary syndrome (PCOS) affects 5-20% of women of reproductive age worldwide. The condition is characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology (PCOM) - with excessive androgen production by the ovaries being a key feature of PCOS. Metabolic dysfunction characterized by insulin resistance and compensatory hyperinsulinaemia is evident in the vast majority of affected individuals. PCOS increases the risk for type 2 diabetes mellitus, gestational diabetes and other pregnancy-related complications, venous thromboembolism, cerebrovascular and cardiovascular events and endometrial cancer. PCOS is a diagnosis of exclusion, based primarily on the presence of hyperandrogenism, ovulatory dysfunction and PCOM. Treatment should be tailored to the complaints and needs of the patient and involves targeting metabolic abnormalities through lifestyle changes, medication and potentially surgery for the prevention and management of excess weight, androgen suppression and/or blockade, endometrial protection, reproductive therapy and the detection and treatment of psychological features. This Primer summarizes the current state of knowledge regarding the epidemiology, mechanisms and pathophysiology, diagnosis, screening and prevention, management and future investigational directions of the disorder.Robert J Norman, Ruijin Wu and Marcin T Stankiewic

Thyrotropin regulation of thyroid peroxidase messenger ribonucleic acid levels in cultured rat thyroid cells: evidence for the involvement of a nontranscriptional mechanism.

The influence of TSH on thyroid peroxidase (TPO) gene expression was investigated in FRTL5 rat thyroid cells. Cultured in the presence of TSH, these cells express a TPO mRNA species of 3.1 kilobases. TSH withdrawal from the culture medium led to a decline in TPO mRNA levels over 24 h. In contrast, no decline in β-actin mRNA levels occurred after 24 h of incubation in TSH-free medium. TSH (1 mU/ml) added to FRTL5 cells cultured in the absence of TSH increased TPO mRNA levels 7- to 9-fold compared to levels in control cells. This effect of TSH on TPO mRNA accumulation in FRTL5 cells was time related (it was already present after 4 h and was maximal after 24 h of cell exposure to TSH), dose related (0.01 and 1 mU/ml were, respectively, the lowest and the maximally effective doses), and independent of new protein synthesis, in that it was not prevented by cycloheximide (100 μM). cAMP analogues [8-bromo-cAMP and (Bu)2cAMP] also increased TPO mRNA levels, although to a lesser degree than TSH. Run-on transcription analysis in nuclei prepared from FRTL5 cells previously cultured in the presence or absence of TSH did not reveal any difference in TPO mRNA transcripts. These results suggest that TSH regulatese the level of TPO mRNA in FRTL5 cells, in part via the second messenger cAMP and by a nontranscriptional mechanism. This TSH effect may represent a primary site of TSH action in regulating TPO bioactivity

Both the 5' and 3' noncoding regions of the thyrotropin receptor messenger ribonucleic acid influence the level of receptor protein expression in transfected mammalian cells

The molecular basis for the difference in the bioresponsiveness of TSH receptor cell lines from two different laboratories has been investigated. We modified our 4-kb TSH receptor complementary DNA (cDNA) by deleting either the 5' untranslated region (UTR), the 3'UTR, or both UTRs. The 5'UTR contains two false AUG initiation codons followed by a stop codon. The cDNAs in the eukaryotic expression vector pSV2-NEO-ECE, as well as the 5'3'UTR-truncated cDNA in pSVL, were stably transfected into Chinese hamster ovary cells. Pools of more than 100 colonies were studied in order to minimize insertion site-dependent variation in the level of expression. Scatchard analysis of TSH binding indicated that the number of receptors on the surface of Chinese hamster ovary cells expressing the wild-type transcript (similar to 16,000/cell) increased approximately a-fold with 5'UTR deletion, approximately 5-fold with 3'UTR deletion, and approximately 10-fold with both 5'UTR and 3'UTR deletion. TSH binding affinities of all constructs were in the range of 2-5 x 10(-10) M. No significant difference was evident between the 5'3'UTR truncated cDNAs in the two different vectors, pSV2-NEO-ECE and pSVL. The increase in the amplitude of the cAMP response to TSH stimulation was commensurate with the number of receptors expressed on the surface of the different cell lines. Truncation of the 5'UTR did not alter TSH receptor messenger RNA (mRNA) levels relative to the wild-type mRNA. In contrast, the level of the 3'UTR-truncated transcript, as well as the 5'3'UTR-deleted transcript, increased approximately 4-fold independent of the expression vector used. In summary, both the 5'UTR and 3'UTR of the human TSH receptor mRNA influence the level of receptor expression on transfected mammalian cells. In particular, the 3'UTR has a destabilizing influence on the mRNA. These data explain the greater level of TSH receptor expression in cell lines that are transfected with cDNA lacking these regions of the mRNA transcript