Bruce M. Raaka

Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates

Development of the endocrine pancreas includes a series of early events wherein precursor cells cluster, that is migrate to form cell aggregates, which subsequently differentiate into islets of Langerhans. We show that PANC-1 cells, a human pancreatic cell line, differentiates into hormone-producing islet-like cell aggregates after exposure to a defined serum-free medium. These cells were used to provide the following evidence that fibroblast growth factor (FGF)2 is a paracrine chemoattractant during PANC-1 cell clustering: (i) FGF2 is secreted and remains bound to the extracellular matrix from where it may diffuse to form chemoattractive gradients; (ii) a subset of cells expresses FGF receptors (FGFRs) -1, -2, -3, and -4; (iii) inhibition of FGFR tyrosine kinase inhibits cell clustering; and (iv) FGF2 neutralizing antibody inhibits clustering. In addition, adult human islet-derived precursor cells, which cluster and differentiate in a manner similar to PANC-1 cells, also secrete FGF2 and express FGFRs. We conclude that FGF2, acting as a paracrine chemoattractant, stimulates clustering of precursor cells, an early step leading to islet-like cell aggregate formation. Similar processes may occur during development of the islet of Langerhans in humans.

The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation.

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

Discovery of novel Agonists and antagonists of the free fatty acid receptor 1 (FFAR1) using virtual screening

The G-protein-coupled receptor free fatty acid receptor 1 (FFAR1), previously named GPR40, is a possible novel target for the treatment of type 2 diabetes. In an attempt to identify new ligands for this receptor, we performed virtual screening (VS) based on two-dimensional (2D) similarity, three-dimensional (3D) pharmacophore searches, and docking studies by using the structure of known agonists and our model of the ligand binding site, which was validated by mutagenesis. VS of a database of 2.6 million compounds followed by extraction of structural neighbors of functionally confirmed hits resulted in identification of 15 compounds active at FFAR1 either as full agonists, partial agonists, or pure antagonists. Site-directed mutagenesis and docking studies revealed different patterns of ligand-receptor interactions and provided important information on the role of specific amino acids in binding and activation of FFAR1.

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.

Discovery of diverse thyroid hormone receptor antagonists by high-throughput docking

Treatment of hyperthyroidism, a common clinical condition that can have serious manifestations in the elderly, has remained essentially unchanged for >30 years. Directly antagonizing the effect of the thyroid hormone at the receptor level may be a significant improvement for the treatment of hyperthyroid patients. We built a computer model of the thyroid hormone receptor (TR) ligand-binding domain in its predicted antagonist-bound conformation and used a virtual screening algorithm to select 100 TR antagonist candidates out of a library of >250,000 compounds. We were able to obtain 75 of the compounds selected in silico and studied their ability to act as antagonists by using cultured cells that express TR. Fourteen of these compounds were found to antagonize the effect of T3 on TR with IC50s ranging from 1.5 to 30 μM. A small virtual library of compounds, derived from the highest affinity antagonist (1-850) that could be rapidly synthesized, was generated. A second round of virtual screening identified new compounds with predicted increased antagonist activity. These second generation compounds were synthesized, and their ability to act as TR antagonists was confirmed by transfection and receptor binding experiments. The extreme structural diversity of the antagonist compounds shows how receptor-based virtual screening can identify diverse chemistries that comply with the structural rules of TR antagonism.

Human Islet‐Derived Precursor Cells Are Mesenchymal Stromal Cells That Differentiate and Mature to Hormone‐Expressing Cells In Vivo

Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin‐producing cells. Previously, we described proliferative populations of human islet‐derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000‐fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C‐peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105+/CD73+/CD90+ cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas‐derived MSC that are capable of differentiating into hormone‐expressing cells. Their ability to mature into functional insulin‐secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet‐like cells for replacement therapy in type 1 diabetes.

Perilipin family members preferentially sequester to either triacylglycerol-specific or cholesteryl-ester-specific intracellular lipid storage droplets

Summary Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes have distinct Plin gene members and additional protein forms derived from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to different types of lipid droplets that are composed of either triacylcerides or cholesterol esters. Furthermore, Plins with strong association preferences to triacylceride (or cholesterol ester) droplets can re-direct the relative intracellular triacylceride–cholesterol ester balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.

Identification of residues important for agonist recognition and activation in GPR40

GPR40 was formerly an orphan G protein-coupled receptor whose endogenous ligands have recently been identified as free fatty acids (FFAs). The receptor, now named FFA receptor 1, has been implicated in the pathophysiology of type 2 diabetes and is a drug target because of its role in FFA-mediated enhancement of glucose-stimulated insulin release. Guided by molecular modeling, we investigated the molecular determinants contributing to binding of linoleic acid, a C18 polyunsaturated FFA, and GW9508, a synthetic small molecule agonist. Twelve residues within the putative GPR40-binding pocket including hydrophilic/positively charged, aromatic, and hydrophobic residues were identified and were subjected to site-directed mutagenesis. Our results suggest that linoleic acid and GW9508 are anchored on their carboxylate groups by Arg183, Asn244, and Arg258. Moreover, His86, Tyr91, and His137 may contribute to aromatic and/or hydrophobic interactions with GW9508 that are not present, or relatively weak, with linoleic acid. The anchor residues, as well as the residues Tyr12, Tyr91, His137, and Leu186, appear to be important for receptor activation also. Interestingly, His137 and particularly His86 may interact with GW9508 in a manner dependent on its protonation status. The greater number of putative interactions between GPR40 and GW9508 compared with linoleic acid may explain the higher potency of GW9508.

The Ligand-Binding Domains of the Thyroid Hormone/Retinoid Receptor Gene Subfamily Function In Vivo To Mediate Heterodimerization, Gene Silencing, and Transactivation

The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.

A Low Molecular Weight Agonist Signals by Binding to the Transmembrane Domain of Thyroid-stimulating Hormone Receptor (TSHR) and Luteinizing Hormone/Chorionic Gonadotropin Receptor (LHCGR)

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.