Receptor-specific Interactions Research Articles

Development of hyaluronate tethered magnetic nanoparticlesfor targeted anti-cancer drug delivery

Despite the tremendous progress in understanding the molecular basis of the disease, cancer still remains one of the leading causes of deaths. Recently, advances in nanotechnology are rapidly enabling the development of novel, multifunctional materials with combined cancer specific targeting, therapeutic and diagnostic functions within a single nanocomplex (NP) that address the shortcomings of traditional disease diagnostic and therapeutic agents. Among the myriad of nanocarriers, magnetic nanoparticles (MNPs) have sparked extensive promise as novel theranostic applications as these MNPs can be directly targeted to the diseased cells with effective therapeutic efficiency. For this, these MNPs should be modified with some highly biocompatible polymers (specially polysaccharides) exhibiting the cancer targeting properties that can strongly interact with receptors expressed on the target cancer to facilitate accurate detection of the specific cancer and enhanced delivery to the target site while reducing unintended tside effects. Over the last few years, many groups have reported hyaluronic acid (HA) as the targeting agent as it directly delivers targeted MNPs to CD44 overexpressed cancer cells. In most of the cases, doxorubicin (DOX) has been used as the anticancer drug as it is largely utilized for treating a broad spectrum of cancers. In our work, we have designed a novel, intravenously injectable, CD44 receptor targeted MNP formulation, where the HA moiety of MNPs facilitate easy detection of cancer cells via receptor specific interactions, DOX can regress the cancer cells with simultaneous imaging efficacy. This theranostic MNPs led to the formation of novel nanoformulation, capable of performing concomitant detection, regression and imaging in in vitro CD44 over expressing cancer cells.

Recent Advances in Mucoadhesive Interface Materials, Mucoadhesion Characterization, and Technologies

AbstractMucoadhesion is an extremely important field of adhesion science and the comprehensive understanding and modulation of mucoadhesion can lead to lifesaving materials and technologies. For instance, deadly cases of COVID‐19 (SARS‐CoV‐2) cytokine storm are associated with viral adhesion and overproduction of mucus, which obstructs the airways. Mucin is the key polymeric compound that is known as a family of high molecular weight, heavily glycosylated proteins in epithelial tissues. Mucoadhesion can occur in many different ways such as receptor specific and charge interactions, covalent or noncovalent bonds. New mucin‐mimic polymers that replicate its beneficial traits can prevent biofilm formation and biofouling not only in biotechnology but also in membrane technologies. This review addresses the latest understandings related to mucin's role in wet adhesion considering different physiological conditions and shows how this translates into interfacial polymer adhesion. Advances in mucoadhesion measurement techniques including the rheological aspects of polymer–mucin adhesive interactions are presented. Specific mucoadhesive systems are discussed such as hydrogel mucoadhesion, catechol/dopamine functionalization, and polymeric nanoparticles. This overview may expand the current understanding of mucoadhesion between soft materials but also contributes to elastocapillary phenomena in soft materials design and applications such as new membranes, drugs, pharmaceutical devices, and lubricated surfaces.

Glycosylation of PAMAM dendrimers significantly improves tumor macrophage targeting and specificity in glioblastoma

Glioblastoma is among the most aggressive forms of cancers, with a median survival of just 15–20 months for patients despite maximum clinical intervention. The majority of conventional anti-cancer therapies fail due to associated off-site toxicities which can be addressed by developing target-specific drug delivery systems. Advances in nanotechnology have provided targeted systems to overcome drug delivery barriers associated with brain and other types of cancers. Dendrimers have emerged as promising vehicles for targeted drug and gene delivery. Dendrimer-mediated targeting strategies can be further enhanced through the addition of targeting ligands to enable receptor-specific interactions. Here, we explore the sugar moieties as ligands conjugated to hydroxyl-terminated polyamidoamine dendrimers to leverage altered metabolism in cancer and immune targeting. Using a highly facile click chemistry approach, we modified the surface of dendrimers with glucose, mannose, or galactose moieties in a well-defined manner, to target upregulated sugar transporters in the context of glioblastoma. We show that glucose modification significantly enhanced targeting of tumor-associated macrophages (TAMs) and microglia by increasing brain penetration and cellular internalization, while galactose modification shifts targeting away from TAMs towards galectins on glioblastoma tumor cells. Mannose modification did not alter TAMs and microglia targeting of these dendrimers, but did alter their kinetics of accumulation within the GBM tumor. The whole body biodistribution was largely similar between the systems. These results demonstrate that dendrimers are versatile delivery vehicles that can be modified to tailor their targeting for the treatment of glioblastoma and other cancers.

Structural Perspective on Ancient Neuropeptide Y-like System reveals Hallmark Features for Peptide Recognition and Receptor Activation

The neuropeptide Y (NPY) family is a peptide-activated G protein-coupled receptor system conserved across all bilaterians, and is involved in food intake, learning, and behavior. We hypothesized that comparing the NPY system in evolutionarily ancient organisms can reveal structural determinants of peptide recognition and receptor activation conserved in evolution. To test this hypothesis, we investigated the homologous FLP/NPR system of the protostome C.elegans. For three prototypic peptide–receptor complexes representing different ligand types, we integrate extensive functional data into structural models of the receptors. Common features include acidic patches in the extracellular loops (ECLs) of the receptors that cooperatively ‘draw’ the peptide into the binding pocket, which was functionally validated in vivo. A structurally conserved glutamate in the ECL2 anchors the peptides by a conserved salt bridge to the arginine of the RFamide motif. Beyond this conserved interaction, peptide binding show variability enabled by receptor-specific interactions. The family-conserved residue Q3.32 is a key player for peptide binding and receptor activation. Altered interaction patterns at Q3.32 may drastically increase the efficacy to activate the receptor.

Surface Modifications for Improved Delivery and Function of Therapeutic Bacteria.

Live therapeutic bacteria (LTBs) hold promise to treat microbiome-related diseases. However, few approaches to improve the colonization of LTBs in the gastrointestinal tract exist, despite colonization being a prerequisite for efficacy of many LTBs. Here, a modular platform to rapidly modify the surface of LTBs to enable receptor-specific interactions with target surfaces is reported. Inspired by bacterial adhesins that facilitate colonization, synthetic adhesins (SAs) are developed for LTBs in the form of antibodies conjugated to their surface. The SA platform is nontoxic, does not alter LTB growth kinetics, and can be used with any antibody or bacterial strain combination. By improving adhesion, SA-modified bacteria demonstrate enhanced in vitro pathogen exclusion from cell monolayers. In vivo kinetics of SA-modified LTBs is tracked in the feces and intestines of treated mice, demonstrating that SA-modified bacteria alter short-term intestinal transit and improve LTB colonization and pharmacokinetics. This platform enables rapid formation of an intestinal niche, leading to an increased maximum concentration and a 20% improvement in total LTB exposure. This work is the first application of traditional pharmacokinetic analysis to design and evaluate LTB drug delivery systems and provides a platform toward controlling adhesion, colonization, and efficacy of LTBs.

Targeting the C-type lectins-mediated host-pathogen interactions with dextran.

Dextran, the α-1,6-linked glucose polymer widely used in biology and medicine, promises new applications. Linear dextran applied as a blood plasma substitute demonstrates a high rate of biocompatibility. Dextran is present in foods, drugs, and vaccines and in most cases is applied as a biologically inert substance. In this review we analyze dextran's cellular uptake principles, receptor specificity and, therefore, its ability to interfere with pathogen-lectin interactions: a promising basis for new antimicrobial strategies. Dextran-binding receptors in humans include the DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) family receptors: DC-SIGN (CD209) and L-SIGN (the liver and lymphatic endothelium homologue of DC-SIGN), the mannose receptor (CD206), and langerin. These receptors take part in the uptake of pathogens by dendritic cells and macrophages and may also participate in the modulation of immune responses, mostly shown to be beneficial for pathogens per se rather than host(s). It is logical to predict that owing to receptor-specific interactions, dextran or its derivatives can interfere with these immune responses and improve infection outcome. Recent data support this hypothesis. We consider dextran a promising molecule for the development of lectin-glycan interaction-blocking molecules (such as DC-SIGN inhibitors) that could be applied in the treatment of diseases including tuberculosis, influenza, hepatitis B and C, human immunodeficiency virus infection and AIDS, etc. Dextran derivatives indeed change the pathology of infections dependent on DC-SIGN and mannose receptors. Complete knowledge of specific dextran-lectin interactions may also be important for development of future dextran applications in biological research and medicine.

Abstract PR5: Treatment of ovarian cancer with targeted tumor-penetrating siRNA nanocomplexes

Abstract Whole-genome analysis of cancer samples is identifying many potential therapeutic targets, by virtue of their being frequently mutated or functionally essential in specific types of cancer. However, we lack efficient ways to test the therapeutic benefit of modulating targets in vivo. RNAi offers one potential solution; however, approaches to deliver siRNA in vivo have been challenging due to their susceptibility to serum nucleases, endosomal entrapment, and stimulation of innate immunity. Furthermore, nanoparticle- and antibody-based siRNA delivery approaches have historically suffered from limited tumor penetration and low transvascular transit, thereby limiting the applicability of parenchymal siRNA targets. Here we describe a tumor penetrating nanocomplex (TPN) comprised of siRNA complexed to a tandem tumor-penetrating and membrane-translocating peptide, which enables the homing of siRNA deep into tumor parenchyma. Upon complexation with siRNA, the resulting nanocomplex is stable, non-immunostimulatory, displays homing peptides in a multivalent fashion that increases their binding avidity and delivers siRNA to the cytosol of tumor cells through receptor-specific interactions and membrane translocation. Upon systemic administration into mice, this nanocomplex penetrates into the parenchyma of metastatic peritoneal tumors and silences target genes in cells of interest in a receptor-specific manner. We employed TPNs in vivo to evaluate ID4, a novel candidate oncogene in ovarian cancer, which we identified by combining genome-scale RNAi screening of cancer cell lines with genome-scale sequence analysis of patient tumors. We show that treatment of tumor-bearing mice with ID4-specific TPNs suppresses tumor growth and significantly improved survival. These findings provide a framework for the identification, credentialing, and understanding of novel cancer targets as well as validating a specific therapeutic target in ovarian cancer. This abstract is also presented as Poster B2. Citation Format: Yin Ren, Hiu Wing Cheung, Ronny Drapkin, David Root, Justin Lo, Valentina Fogal, Erkki Ruoslahti, William Hahn, Sangeeta Bhatia, Geoffrey von Maltzahn, Amit Agrawal, Glenn Cowley, Barbara Weir, Jesse Boehm, Pablo Tamayo, Jill Mesirov, Alison Karst. Treatment of ovarian cancer with targeted tumor-penetrating siRNA nanocomplexes [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr PR5.

Molecular basis of signaling specificity of insulin and IGF receptors: neglected corners and recent advances.

Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of “metabolic” and “mitogenic” responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to “metabolic” and “mitogenic” responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in “metabolic” or “mitogenic” signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears to be the major determinant of whether responses to insulin and IGFs are perceived as “metabolic” or “mitogenic.”

The impact of oral vitamin A derivatives on lipid metabolism – What recommendations can be derived for dealing with this issue in the daily dermatological practice?

Retinoids and vitamin A derivatives have been widely used topically and systemically in the treatment of hyper- and parakeratotic skin diseases, genodermatoses, severe acne, autoimmune diseases (i. e. lupus erythematosus) or cutaneous T-cell lymphoma for more than 30 years. In addition to the desired proliferation-inhibiting, differentiation-inducing and antiinflammatory or sebo-suppressive effects, vitamin A derivatives also affect lipid metabolism. This is shown primarily by an increase of transaminases, triglycerides or cholesterol levels which vary in intensity from patient to patient. The degree of impact on the different parameters of lipid metabolism depends on the nature of the vitamin A derivative on the one hand due to different receptor specific binding interactions (RAR/RXR), while on the other hand posttranslational processes also play a major role. This review paper gives a brief, concise overview of the vitamin A derivatives and possible effects on lipid metabolism that can be expected. Additionally it contains a recommendation for secure handling of abnormal laboratory values before, during and after oral therapy with vitamin A derivatives. The aim of this article is to provide practical help and confidence in dealing with vitamin A derivatives in daily clinical practice. The publication was created in cooperation with the Deutsche Dermatologische Gesellschaft (DDG) and Deutsche Gesellschaft zur Bekämpfung von Fettstoffwechselstörungen und ihren Folgeerkrankungen (DGFF [Lipid-Liga] e. V.).

Human Airway Epithelial Cell Responses to Neisseria lactamica and Purified Porin via Toll-Like Receptor 2-Dependent Signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin's surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Receptor and Subunit Specific Interactions of RIC-3 with Nicotinic Acetylcholine Receptors

RIC-3 belongs to a conserved family of proteins influencing maturation of nicotinic acetylcholine receptors (nAChRs). RIC-3 homologues were shown to differently affect different nAChRs. Here we show that coexpression with RIC-3 increases the level of surface expression of DEG-3 while slightly reducing the level of surface expression of DES-2, both subunits of the DEG-3/DES-2 nAChRs. Those different effects are a likely explanation for the previously demonstrated effects of RIC-3, an endoplasmic reticulum resident protein, on properties of this receptor. To understand how RIC-3 interacts with different nAChR subunits, we identified and characterized domains and residues enabling this interaction. This analysis shows that conserved residues in the second RIC-3 transmembrane domain are needed for its interactions with two different Caenorhabditis elegans nAChRs, DEG-3/DES-2 and ACR-16. These conserved residues do not, however, function alone; rather, we show that additional domains also enable RIC-3's interactions with these receptors. Interestingly, the relative importance of these residues or of other domains mediating interactions of RIC-3 with nAChRs differs for the two different receptors. Differences in the way that RIC-3, predicted to be an intrinsically disordered protein, interacts with different receptors and receptor subunits suggest that it may adopt different conformations to enable these interactions. Such differences may explain both the effects of RIC-3 on receptor properties and the differences in its effects on different receptors.

Gastrin releasing protein receptor specific gold nanorods: breast and prostate tumor avid nanovectors for molecular imaging.

Gastrin releasing protein receptor specific bombesin (BBN) peptide-gold nanoconjugates were successfully synthesized using gold nanorods and dithiolated peptide. The gold nanorod-bombesin (GNR-BBN) conjugates showed extraordinary in vitro stabilities against various biomolecules including NaCl, cysteine, histidine, bovine serum albumin, human serum albumin, and dithiothreitol. Quantitative measurements on the binding affinity (IC(50)) of GNR-BBN conjugates toward prostate and breast tumor cells were evaluated. The IC(50) values establish that GNR-BBN conjugates have strong affinity toward the gastrin releasing peptide receptors on both the tumors. Detailed cellular interaction studies of GNR-BBN conjugates revealed that nanorods internalize via a receptor-mediated endocytosis pathway. The receptor specific interactions of GNR-BBN conjugates provide realistic opportunities in the design and development of in vivo molecular imaging and therapy agents for cancer.

The conserved RIC-3 coiled-coil domain mediates receptor-specific interactions with nicotinic acetylcholine receptors.

RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation.

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FN's central cell-binding domains (FN III9–10) affects its integrin specificity. Through production of FN III9–10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin α 5β 1 and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of α 5β 1 integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that α 5β 1 has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for α 5β 1 versus α vβ 3 results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.

Efficacy and Mechanisms of α-Solasonine-and α-Solamargine-Induced Cytolysis on Two Strains of Trypanosoma cruzi

Two genetically diverse strains of Trypanosoma cruzi were tested in vitro for susceptibility to the solanum-derived triglycoside alkaloids solasonine and solamargine. Cytolytic assays were performed on epimastigotes (EMs) and bloodstream form trypomastigotes (BSFs) lifecycle stages by using serial dilutions of each alkaloid. Each alkaloid effectively lysed both lifecycle stages, although solasonine routinely required higher concentrations to induce similar results. EMs demonstrated greater resistance to cytolysis than BSFs at equal concentrations of either alkaloid. No significant resistance could be correlated to parasite strain. The reported synergistic cytolytic effects observed upon compounding solasonine and solamargine together were also tested. We failed to identify any cytolytic synergism in cultures of EMs or BSFs. The role of rhamnose-binding proteins (RBPs) in mediating cytolysis was investigated through competitive inhibition experiments. The addition of exogenous L: -rhamnose to the media failed to reduce parasite attrition independent of the parasite lifecycle stage. Based on these results, we suggest the mechanisms involved in cytolysis of T. cruzi by solasonine and solamargine are largely independent of rhamnose receptor-specific interactions. We propose that attrition likely involves less-specific carbohydrate interactions, which lead to the formation and intercalation of sterol complexes into the parasite plasma membrane.

Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids.

Coregulator Interactions with the Thyroid Hormone Receptor

The thyroid hormone receptor (TR) directly regulates the transcription of thyroid hormone-responsive genes in response to changing levels of thyroid hormone. Mechanistically TR utilizes a complex set of binding interactions, with hormone, response elements, and coregulatory proteins, to provide specific local control of patterns of transcriptional response that are partially responsible for inducing the tissue-selective responses to the circulating hormone. One of the apparently dominant phenomena in the regulation of thyroid hormone responses is the protein interactions between TR and its coregulators. This review summarizes the current state of knowledge with respect to the identity of these coregulators, their interaction with TR, and the consequences of those interactions.

The manifold actions of the protein inhibitor of activated STAT proteins on the transcriptional activity of mineralocorticoid and glucocorticoid receptors in neural cells.

Corticosteroid actions in the brain are exerted via the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). These receptors share several structural and functional similarities but their activation in the brain triggers distinct biological actions, for instance on neuronal survival or the regulation of the hypothalamo-pituitary-adrenal axis. Like other hormone-activated receptors, the transcriptional properties of the MR and GR depend on their ability to recruit a variety of co-regulators, which modulate their activity on target promoters, in a specific manner. The N-terminal regions of the MR and GR share the smallest degree of sequence conservation, whereas they display opposite effects on the transactivation properties of these receptors; thus, they may provide surfaces suitable for receptorspecific interactions with co-regulatory proteins. Here, we employed a yeast two-hybrid system to identify molecules interacting with the N-terminal part of the MR (amino acids 170-433). This approach resulted in the isolation of representative cDNAs from all members of the protein inhibitor of activated STAT (PIAS) family of proteins as potential MR-interacting partners. In neural cells, PIAS3 exhibited a strong and specific interaction with MR, but not GR, as indicated by mammalian two-hybrid assays and co-immunoprecipitation experiments in vivo. The interaction with MR was enhanced in the presence of aldosterone, an MR agonist, and was found to occur through a conserved, serine- and acidic amino acid residue-rich domain of PIAS3. To compare the modulatory properties of PIAS proteins on MR and GR transcriptional activity in a neural environment, MMTV reporter gene assays were performed in the human neuroblastoma cell line SK-N-MC. This analysis revealed that PIAS3 can inhibit MR, but not GR, transactivation in response to their corresponding ligands. Further, it showed that PIAS1 and PIASxbeta, but not PIASy, could also inhibit MR-mediated transcription despite the lack of detected physical interaction with MR. Interestingly, PIASxbeta and PIASy dose-dependently co-activated GR, whereas PIAS1 impaired GR-induced transcription. Taken together the results reveal differential modulatory roles of the PIAS proteins on the transcriptional properties of MR and GR, thus providing new insights into the bifurcating actions of these two receptors in neural cells where they are frequently co-localized.

The effect of hydrogel charge density on cell attachment

The competitive growth patterns of osteoblasts and fibroblasts can determine if healthy bone or pathologic scar tissue is formed at a wound site. Cell interactions with various alloplastic biomaterials used for tissue-engineering applications is complex. Defined synthetic mediums are valuable for studying ionic and cell receptor–specific interactions. The objectives of this study were to determine if fibroblasts and osteoblasts differentially attached to HEMA and PEG hydrogels copolymerized with positive, negative, or neutral charge densities, or when grafted with specific integrin receptor RGD adhesion ligand. Cytoskeletal phenotypes were assessed with immunofluorescent microscopy and cell attachment assays. Osteoblast cell attachment to both HEMA and PEG hydrogels was significantly higher ( P<0.01) as compared to fibroblast cells. Positively charged HEMA and PEG hydrogels supported the greatest cell attachment, followed by RGD grafted, negative, and neutral charge densities, respectively. Each of these conditions elicited nearly a two-fold increase in osteoblast cell attachment, as compared to fibroblasts. Cell attachment to serum-coated coverslips was used as the control. Immunofluorescent analysis showed that both cell types attached and spread better on the positively charged hydrogels. However, fibroblasts demonstrated less spreading as compared to osteoblasts. In conclusion, differences in hydrophilic properties differentially affect osteoblast and fibroblast cell attachment and spreading.

Thyroid hormone receptor-specific interactions with steroid receptor coactivator-1 in the pituitary.

Steroid receptor coactivator-1 (SRC-1) is a transcription cofactor that enhances the hormone-dependent action mediated by the thyroid hormone (TH) receptor (TR) as well as other nuclear receptors. However, it is not known whether the SRC-1-mediated activation of TH-regulated gene transcription is TR isoform specific in the pituitary. We generated mice that were deficient in TRalpha and SRC-1 (TRalpha(0/0)SRC-1(-/-)), as well in TRbeta and SRC-1 (TRbeta(-/-)SRC-1(-/-)), and thyroid function tests and effects of TH deprivation and TH treatment were compared with wild-type mice or mice with deletion of either TRs or SRC-1 alone. We have shown that 1) TRbeta(-/-)SRC-1(-/-) mice demonstrate more severe TH resistance than either the SRC-1(-/-) or TRbeta(-/-) mice; the additive effect indicates that SRC-1 has an independent role in TH action over that of TRbeta; 2) SRC-1 facilitates TRbeta and TRalpha-mediated down-regulation of TSH, as TRalpha(0/0)SRC-1(-/-) mice demonstrate TH resistance rather than hypersensitivity as seen in TRalpha(0/0)mice; and 3) a compensatory increase in SRC-1 expression is associated with the TH hypersensitivity seen in TRalpha-deficient animals. We conclude that SRC-1 action in the pituitary mediates TH action via specific TR subtypes.