Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling

Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investigated the roles of these domains in the function of SSI-1. Results of reporter assays demonstrated that the pre-SH2 domain (24 aa in front of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression studies of COS7 cells revealed that these domains also were required for inhibition of three JAKs (JAK1, JAK2, and TYK2). Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of association of SSI-1 with TYK2. Thus, SSI-1 associates with JAK family kinase via its SH2 domain, and the pre-SH2 domain is required for the function of SSI-1. Deletion of the SC-motif markedly reduced expression of SSI-1 protein in M1 cells, and this reduction was reversed by treatment with proteasome inhibitors, suggesting that this motif is required to protect the SSI-1 molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the suppression of interleukin 6 signaling.

Identification and characterization of a retinoid-induced class II tumor suppressor/growth regulatory gene

Retinoids, synthetic and natural analogs of retinoic acid, exhibit potent growth inhibitory and cell differentiation activities that account for their beneficial effects in treating hyperproliferative diseases such as psoriasis, actinic keratosis, and certain neoplasias. Tazarotene is a synthetic retinoid that is used in the clinic for the treatment of psoriasis. To better understand the mechanism of retinoid action in the treatment of hyperproliferative diseases, we used a long-range differential display–PCR to isolate retinoid-responsive genes from primary human keratinocytes. We have identified a cDNA, tazarotene-induced gene 3 (TIG3; Retinoic Acid Receptor Responder 3) showing significant homology to the class II tumor suppressor gene, H-rev 107. Tazarotene treatment increases TIG3 expression in primary human keratinocytes and in vivo in psoriatic lesions. Increased TIG3 expression is correlated with decreased proliferation. TIG3 is expressed in a number of tissues, and expression is reduced in cancer cell lines and some primary tumors. In breast cancer cell lines, retinoid-dependent TIG3 induction is observed in lines that are growth suppressed by retinoids but not in nonresponsive lines. Transient over-expression of TIG3 in T47D or Chinese hamster ovary cells inhibits colony expansion. Finally, studies in 293 cells expressing TIG3 linked to an inducible promoter demonstrated decreased proliferation with increased TIG3 levels. These studies suggest that TIG3 may be a growth regulator that mediates some of the growth suppressive effects of retinoids.

Acute leukemia with promyelocytic features in PML/RARα transgenic mice

Acute promyelocytic leukemia (APL) is associated with reciprocal chromosomal translocations involving the retinoic acid receptor α (RARα) locus on chromosome 17. In the majority of cases, RARα translocates and fuses with the promyelocytic leukemia (PML) gene located on chromosome 15. The resulting fusion genes encode the two structurally unique PML/RARα and RARα/PML fusion proteins as well as aberrant PML gene products, the respective pathogenetic roles of which have not been elucidated. We have generated transgenic mice in which the PML/RARα fusion protein is specifically expressed in the myeloid-promyelocytic lineage. During their first year of life, all the PML/RARα transgenic mice have an abnormal hematopoiesis that can best be described as a myeloproliferative disorder. Between 12 and 14 months of age, 10% of them develop a form of acute leukemia with a differentiation block at the promyelocytic stage that closely mimics human APL even in its response to retinoic acid. Our results are conclusive in vivo evidence that PML/RARα plays a crucial role in the pathogenesis of APL.

The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ± 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% ± 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β2-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β2-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β2-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (i) is retained in the endoplasmic reticulum and middle Golgi compartment, (ii) fails to undergo late Golgi processing, and (iii) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.

Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

Molecular identification of human G-substrate, a possible downstream component of the cGMP-dependent protein kinase cascade in cerebellar Purkinje cells

G-substrate, an endogenous substrate for cGMP-dependent protein kinase, exists almost exclusively in cerebellar Purkinje cells, where it is possibly involved in the induction of long-term depression. A G-substrate cDNA was identified by screening expressed sequence tag databases from a human brain library. The deduced amino acid sequence of human G-substrate contained two putative phosphorylation sites (Thr-68 and Thr-119) with amino acid sequences [KPRRKDT(p)PALH] that were identical to those reported for rabbit G-substrate. G-substrate mRNA was expressed almost exclusively in the cerebellum as a single transcript. The human G-substrate gene was mapped to human chromosome 7p15 by radiation hybrid panel analysis. In vitro translation products of the cDNA showed an apparent molecular mass of 24 kDa on SDS/PAGE which was close to that of purified rabbit G-substrate (23 kDa). Bacterially expressed human G-substrate is a heat-stable and acid-soluble protein that cross-reacts with antibodies raised against rabbit G-substrate. Recombinant human G-substrate was phosphorylated efficiently by cGMP-dependent protein kinase exclusively at Thr residues, and it was recognized by antibodies specific for rabbit phospho-G-substrate. The amino acid sequences surrounding the sites of phosphorylation in G-substrate are related to those around Thr-34 and Thr-35 of the dopamine- and cAMP-regulated phosphoprotein DARPP-32 and inhibitor-1, respectively, two potent inhibitors of protein phosphatase 1. However, purified G-substrate phosphorylated by cGMP-dependent protein kinase inhibited protein phosphatase 2A more effectively than protein phosphatase 1, suggesting a distinct role as a protein phosphatase inhibitor.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties

Bile secretion involves the structural and functional interplay of hepatocytes and cholangiocytes, the cells lining the intrahepatic bile ducts. Hepatocytes actively secrete bile acids into the canalicular space and cholangiocytes then transport bile acids in a vectorial manner across their apical and basolateral plasma membranes. The initial step in the transepithelial transport of bile acids across rat cholangiocytes is apical uptake by a Na+-dependent bile acid transporter (ASBT). To date, the molecular basis of the obligate efflux mechanism for extrusion of bile acids across the cholangiocyte basolateral membrane remains unknown. We have identified an exon-2 skipped, alternatively spliced form of ASBT, designated t-ASBT, expressed in rat cholangiocytes, ileum, and kidney. Alternative splicing causes a frameshift that produces a 154-aa protein. Antipeptide antibodies detected the ≈19 kDa t-ASBT polypeptide in rat cholangiocytes, ileum, and kidney. The t-ASBT was specifically localized to the basolateral domain of cholangiocytes. Transport studies in Xenopus oocytes revealed that t-ASBT can function as a bile acid efflux protein. Thus, alternative splicing changes the cellular targeting of ASBT, alters its functional properties, and provides a mechanism for rat cholangiocytes and other bile acid-transporting epithelia to extrude bile acids. Our work represents an example in which a single gene appears to encode via alternative splicing both uptake and obligate efflux carriers in a bile acid-transporting epithelial cell.

Acquired, nonrandom chromosomal abnormalities associated with the development of acute promyelocytic leukemia in transgenic mice

We previously generated a transgenic mouse model for acute promyelocytic leukemia (APL) by expressing the promyelocytic leukemia (PML)–retinoic acid receptor (RARα) cDNA in early myeloid cells. This fusion protein causes a myeloproliferative disease in 100% of animals, but only 15–20% of the animals develop acute leukemia after a long latency period (6–13 months). PML-RARα is therefore necessary, but not sufficient, for APL development. The coexpression of a reciprocal form of the fusion, RARα-PML, increased the likelihood of APL development (55–60%), but did not shorten latency. Together, these results suggested that additional genetic events are required for the development of APL. We therefore evaluated the splenic tumor cells from 18 transgenic mice with APL for evidence of secondary genetic events, by using spectral karyotyping analysis. Interstitial or terminal deletions of the distal region of one copy of chromosome 2 [del(2)] were found in 1/5 tumors expressing PML-RARα, but in 11/13 tumors expressing both PML-RARα and RARα-PML (P < 0.05). Leukemic cells that contained a deletion on chromosome 2 often contained additional chromosomal gains (especially of 15), chromosomal losses (especially of 11 or X/Y), or were tetraploid (P ≤ 0.001). These changes did not commonly occur in nontransgenic littermates, nor in aged transgenic mice that did not develop APL. These results suggest that expression of RARα-PML increases the likelihood of chromosome 2 deletions in APL cells. Deletion 2 appears to predispose APL cells to further chromosomal instability, which may lead to the acquisition of additional changes that provide an advantage to the transformed cells.

Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis

The transcription factor NF-κB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-κB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IκB is degraded by the ubiquitin–proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-κB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin–proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-κB and the subsequent transcription of genes that are regulated by NF-κB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IκBα degradation and NF-κB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin–proteasome pathway and NF-κB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

A yeast genetic system for selecting small molecule inhibitors of protein–protein interactions in nanodroplets

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein–protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein–protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These “nanodroplets” contain defined media, cells, and one or more beads containing ≈100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein–protein interactions in living cells or organisms.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1α,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17β-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor α (TGFα), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Three independent lines of evidence suggest retinoids as causal to schizophrenia

Retinoid dysregulation may be an important factor in the etiology of schizophrenia. This hypothesis is supported by three independent lines of evidence that triangulate on retinoid involvement in schizophrenia: (i) congenital anomalies similar to those caused by retinoid dysfunction are found in schizophrenics and their relatives; (ii) those loci that have been suggestively linked to schizophrenia are also the loci of the genes of the retinoid cascade (convergent loci); and (iii) the transcriptional activation of the dopamine D2 receptor and numerous schizophrenia candidate genes is regulated by retinoic acid. These findings suggest a close causal relationship between retinoids and the underlying pathophysiological defects in schizophrenia. This leads to specific strategies for linkage analyses in schizophrenia. In view of the heterodimeric nature of the retinoid nuclear receptor transcription factors, e.g., retinoid X receptor β at chromosome 6p21.3 and retinoic acid receptor β at 3p24.3, two-locus linkage models incorporating genes of the retinoid cascade and their heterodimeric partners, e.g., peroxisome proliferator-activated receptor α at chromosome 22q12-q13 or nuclear-related receptor 1 at chromosome 2q22-q23, are proposed. New treatment modalities using retinoid analogs to alter the downstream expression of the dopamine receptors and other genes that are targets of retinoid regulation, and that are thought to be involved in schizophrenia, are suggested.