Acinetobacter baumannii Secretes a Bioactive Lipid That Triggers Inflammatory Signaling and Cell Death.

Acinetobacter baumannii is a highly pathogenic Gram-negative bacterium that causes severe infections with very high fatality rates. A. baumannii infection triggers innate as well as adaptive immunity, however, our understanding of the inflammatory factors secreted by A. baumannii that alarm the immune system remains limited. In this study, we report that the lab adapted and clinical strains of A. baumannii secrete an inflammatory bioactive factor which activates TLR2, leading to canonical IRAK4-dependent NF-κB signaling and production of pro-inflammatory cytokines interleukin (IL)-6 and IL-8 and activation of the inflammasome pathway causing pyroptotic cell death. Biochemical fractionation of the A. baumannii culture filtrate revealed the hydrophobic nature of the inflammatory factor. Concordantly, lipase treatment of the culture filtrate or TLR2 inhibition in macrophages abrogated NF-κB activation and cell death induction. Culture filtrates from the LPS- and lipoprotein-deficient A. baumannii mutants retain immuno-stimulatory properties suggesting that a lipid other than these known stimulatory molecules can trigger inflammation during A. baumannii infection. Our results reveal that A. baumannii secretes a previously unappreciated inflammatory bioactive lipid that activates multiple pro-inflammatory signaling pathways and induces cell death in human and murine macrophages.

Shigella ubiquitin ligase IpaH7.8 targets gasdermin D for degradation to prevent pyroptosis and enable infection.

The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.

Outer membrane vesicles containing OmpA induce mitochondrial fragmentation to promote pathogenesis of Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic resistant Gram-negative bacterium that causes life-threatening infections in humans with a very high mortality rate. A. baumannii is an extracellular pathogen with poorly understood virulence mechanisms. Here we report that A. baumannii employs the release of outer membrane vesicles (OMVs) containing the outer membrane protein A (OmpAAb) to promote bacterial pathogenesis and dissemination. OMVs containing OmpAAb are taken up by mammalian cells where they activate the host GTPase dynamin-related protein 1 (DRP1). OmpAAb mediated activation of DRP1 enhances its accumulation on mitochondria that causes mitochondrial fragmentation, elevation in reactive oxygen species (ROS) production and cell death. Loss of DRP1 rescues these phenotypes. Our data show that OmpAAb is sufficient to induce mitochondrial fragmentation and cytotoxicity since its expression in E. coli transfers its pathogenic properties to E. coli. A. baumannii infection in mice also induces mitochondrial damage in alveolar macrophages in an OmpAAb dependent manner. We finally show that OmpAAb is also required for systemic dissemination in the mouse lung infection model. In this study we uncover the mechanism of OmpAAb as a virulence factor in A. baumannii infections and further establish the host cell factor required for its pathogenic effects.

A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase.

UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo.

UNLABELLED: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

Molecular basis for specific recognition of bacterial ligands by NAIP/NLRC4 inflammasomes.

NLR (nucleotide-binding domain [NBD]- and leucine-rich repeat [LRR]-containing) proteins mediate innate immune sensing of pathogens in mammals and plants. How NLRs detect their cognate stimuli remains poorly understood. Here, we analyzed ligand recognition by NLR apoptosis inhibitory protein (NAIP) inflammasomes. Mice express multiple highly related NAIP paralogs that recognize distinct bacterial proteins. We analyzed a panel of 43 chimeric NAIPs, allowing us to map the NAIP domain responsible for specific ligand detection. Surprisingly, ligand specificity was mediated not by the LRR domain, but by an internal region encompassing several NBD-associated α-helical domains. Interestingly, we find that the ligand specificity domain has evolved under positive selection in both rodents and primates. We further show that ligand binding is required for the subsequent co-oligomerization of NAIPs with the downstream signaling adaptor NLR family, CARD-containing 4 (NLRC4). These data provide a molecular basis for how NLRs detect ligands and assemble into inflammasomes.

Blue native polyacrylamide gel electrophoresis to monitor inflammasome assembly and composition.

Inflammasomes are large cytosolic multi-protein complexes that form in response to infection and other stimuli, and play an important role in the innate immune response. Traditional methods for assaying inflammasome activation have relied on detection of autoproteolysis of the cysteine protease caspase-1, or proteolytic processing of its substrate, the inflammatory cytokine IL-1ß. These measurements report on the final output of inflammasome activation, but do not assess inflammasome assembly and composition. Here we describe the application of blue native gel electrophoresis as a tool for investigating stimulus-dependent inflammasome assembly and for identification of individual components within the multi-protein complex.

A versatile, bar-coded nuclear marker/reporter for live cell fluorescent and multiplexed high content imaging.

The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments. However, the number of distinct fluorescent outputs available remains limited. Here, we describe a cellular bar-code technology in which multiple cell-based assays are combined in one well after which each assay is distinguished by fluorescence microscopy. The technology uses the unique fluorescent properties of assay-specific markers comprised of distinct combinations of different 'red' fluorescent proteins sandwiched around a nuclear localization signal. The bar-code markers are excited by a common wavelength of light but distinguished ratiometrically by their differing relative fluorescence in two emission channels. Targeting the bar-code to cell nuclei enables individual cells expressing distinguishable markers to be readily separated by standard image analysis programs. We validated the method by showing that the unique responses of different cell-based assays to specific drugs are retained when three assays are co-plated and separated by the bar-code. Based upon those studies, we discuss a roadmap in which even more assays may be combined in a well. The ability to analyze multiple assays simultaneously will enable screens that better identify, characterize and distinguish hits according to multiple biologically or clinically relevant criteria. These capabilities also enable the re-creation of complex mixtures of cell types that is emerging as a central area of interest in many fields.

Recognition of bacteria by inflammasomes.

Inflammasomes are cytosolic multiprotein complexes that assemble in response to a variety of infectious and noxious insults. Inflammasomes play a critical role in the initiation of innate immune responses, primarily by serving as platforms for the activation of inflammatory caspase proteases. One such caspase, CASPASE-1 (CASP1), initiates innate immune responses by cleaving pro-IL-1ß and pro-IL-18, leading to their activation and release. CASP1 and another inflammatory caspase termed CASP11 can also initiate a rapid and inflammatory form of cell death termed pyroptosis. Several distinct inflammasomes have been described, each of which contains a unique sensor protein of the NLR (nucleotide-binding domain, leucine-rich repeat-containing) superfamily or the PYHIN (PYRIN and HIN-200 domain-containing) superfamily. Here we describe the surprisingly diverse mechanisms by which NLR/PYHIN proteins sense bacteria and initiate innate immune responses. We conclude that inflammasomes represent a highly adaptable scaffold ideally suited for detecting and initiating rapid innate responses to diverse and rapidly evolving bacteria.

NAIPs: building an innate immune barrier against bacterial pathogens. NAIPs function as sensors that initiate innate immunity by detection of bacterial proteins in the host cell cytosol.

The innate immune system of mammals encodes several families of immune detector proteins that monitor the cytosol for signs of pathogen invasion. One important but poorly understood family of cytosolic immunosurveillance proteins is the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins. Recent work has demonstrated that one subfamily of NLRs, the NAIPs (NLR family, apoptosis inhibitory proteins), are activated by specific interaction with bacterial ligands, such as flagellin. NAIP activation leads to assembly of a large multiprotein complex called the inflammasome, which initiates innate immune responses by activation of the Caspase-1 protease. NAIPs therefore appear to detect pathogen molecules via a simple and direct receptor-ligand mechanism. Interestingly, other NLR family members appear to detect pathogens indirectly, perhaps by responding to host cell "stress" caused by the pathogen. Thus, the NLR family may have evolved surprisingly diverse mechanisms for detecting pathogens.

Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity.

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the caspase 1 protease. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing 4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin and PrgJ, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homologue of NLRC4, NAIP5 (NLR family, apoptosis inhibitory protein 5), has been implicated in activation of NLRC4 (refs 7-11), but is widely assumed to have only an auxiliary role, as NAIP5 is often dispensable for NLRC4 activation. However, Naip5 is a member of a small multigene family, raising the possibility of redundancy and functional specialization among Naip genes. Here we show in mice that different NAIP paralogues determine the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that the NAIP2-NLRC4 complex physically associates with PrgJ but not flagellin, whereas NAIP5-NLRC4 associates with flagellin but not PrgJ. Our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP-NLRC4 inflammasomes.

Differential requirements for NAIP5 in activation of the NLRC4 inflammasome.

Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domain-leucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

Structure, affinity, and availability of estrogen receptor complexes in the cellular environment.

An ability to measure the biochemical parameters and structures of protein complexes at defined locations within the cellular environment would improve our understanding of cellular function. We describe widely applicable, calibrated Förster resonance energy transfer methods that quantify structural and biochemical parameters for interaction of the human estrogen receptor alpha-isoform (ER alpha) with the receptor interacting domains (RIDs) of three cofactors (SRC1, SRC2, SRC3) in living cells. The interactions of ER alpha with all three SRC-RIDs, measured throughout the cell nucleus, transitioned from structurally similar, high affinity complexes containing two ER alphas at low free SRC-RID concentrations (<2 nm) to lower affinity complexes with an ER alpha monomer at higher SRC-RID concentrations (approximately 10 nm). The methods also showed that only a subpopulation of ER alpha was available to form complexes with the SRC-RIDs in the cell. These methods represent a template for extracting unprecedented details of the biochemistry and structure of any complex that is capable of being measured by Förster resonance energy transfer in the cellular environment.

Dimerization between aequorea fluorescent proteins does not affect interaction between tagged estrogen receptors in living cells.

Forster resonance energy transfer (FRET) detection of protein interaction in living cells is commonly measured following the expression of interacting proteins genetically fused to the cyan (CFP) and yellow (YFP) derivatives of the Aequorea victoria fluorescent protein (FP). These FPs can dimerize at mM concentrations, which may introduce artifacts into the measurement of interaction between proteins that are fused with the FPs. Here, FRET analysis of the interaction between estrogen receptors (alpha isoform, ERalpha) labeled with "wild-type" CFP and YFP is compared with that of ERalpha labeled with "monomeric" A206K mutants of CFP and YFP. The intracellular equilibrium dissociation constant for the hormone-induced ERalpha-ERalpha interaction is similar for ERalpha labeled with wild-type or monomeric FPs. However, the measurement of energy transfer measured for ERalpha-ERalpha interaction in each cell is less consistent with the monomeric FPs. Thus, dimerization of the FPs does not affect the kinetics of ERalpha-ERalpha interaction but, when brought close together via ERalpha-ERalpha interaction, FP dimerization modestly improves FRET measurement.

Functional sequestration of transcription factor activity by repetitive DNA.

Higher eukaryote genomes contain repetitive DNAs, often concentrated in transcriptionally inactive heterochromatin. Although repetitive DNAs are not typically considered as regulatory elements that directly affect transcription, they can contain binding sites for some transcription factors. Here, we demonstrate that binding of the transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) to the mouse major alpha-satellite repetitive DNA sequesters C/EBPalpha in the transcriptionally inert pericentromeric heterochromatin. We find that this sequestration reduces the transcriptional capacity of C/EBPalpha. Functional sequestration of C/EBPalpha was demonstrated by experimentally reducing C/EBPalpha binding to the major alpha-satellite DNA, which elevated the concentration of C/EBPalpha in the non-heterochromatic subcompartment of the cell nucleus. The reduction in C/EBPalpha binding to alpha-satellite DNA was induced by the co-expression of the transcription factor Pit-1, which removes C/EBPalpha from the heterochromatic compartment, and by the introduction of an altered-specificity mutation into C/EBPalpha that reduces binding to alpha-satellite DNA but permits normal binding to sites in some gene promoters. In both cases the loss of alpha-satellite DNA binding coincided with an elevation in the binding of C/EBPalpha to a promoter and an increased transcriptional output from that promoter. Thus, the binding of C/EBPalpha to this highly repetitive DNA reduced the amount of C/EBPalpha available for binding to and regulation of this promoter. The functional sequestration of some transcription factors through binding to repetitive DNAs may represent an underappreciated mechanism controlling transcription output.

Ligand-selective interdomain conformations of estrogen receptor-alpha.

Selective estrogen receptor modulators (SERMs) inhibit estrogen activation of the estrogen receptor (ER) in some tissues but activate ER in other tissues. These tissue-selective actions suggest that SERMs may be identified with tissue specificities that would improve the safety of breast cancer and hormone replacement therapies. The identification of an improved SERM would be aided by understanding the effects of each SERM on the structure and interactions of ER. To date, the inability to obtain structures of the full-length ER has limited our structural characterization of SERM action to their antiestrogenic effects on the isolated ER ligand binding domain. We studied the effects of estradiol and the clinically useful SERMs 4-hydroxytamoxifen and fulvestrant on the conformation of the full-length ERalpha dimer complex by comparing, in living human breast cancer cells, the amounts of energy transfer between fluorophores attached to different domains of ERalpha. Estradiol, 4-hydroxytamoxifen, and fulvestrant all promoted the rapid formation of ERalpha dimers with equivalent interaction kinetics. The amino- and carboxyl-terminal ERalpha domains both contain activation functions differentially affected by these ligands, but the positions of only the carboxyl termini differed upon binding with estradiol, 4-hydroxytamoxifen, or fulvestrant. The association of a specific ERalpha dimer conformation with the binding of ligands of different clinical effect will assist the identification of a SERM with optimal tissue-selective estrogenic and antiestrogenic activities. These studies also provide a roadmap for dissecting important structural and kinetic details for any protein complex from the quantitative analysis of energy transfer.

A mutant signal transducer and activator of transcription 5b, associated with growth hormone insensitivity and insulin-like growth factor-I deficiency, cannot function as a signal transducer or transcription factor.

CONTEXT: A natural missense mutation in the signal transducer and activator of transcription (STAT) 5b gene was recently identified in association with a female patient presenting with severe growth failure and immune dysfunction. The mutation results in an alanine to proline substitution at residue 630 (A630P) in the src-homology-2 domain, a region essential for docking of STATs to phospho-tyrosines on activated receptors, STAT dimerization, and stabilization of phospho-STAT-DNA interactions. OBJECTIVE: The purpose of this study was to explore the molecular mechanisms underlying the GH insensitivity and IGF-I deficiency caused by the A630P-mutated STAT5b. RESULTS: In reconstitution experiments using HEK293 cells, both GH and interferon-gamma were unable to activate mutant STAT5b (A630P), as demonstrated by lack of immunodetectable phospho-tyrosyl-STAT5b (A630P) and inability to drive luciferase reporter activity. However, the Src family of nonreceptor kinases [constitutively active v-src and epithelial growth factor-induced c-src] tyrosine-phosphorylated STAT5b(A630P). The v-src-induced phospho-STAT5b(A630P) translocated to the nucleus but, unlike wild-type Stat5b, was unable to bind DNA. CONCLUSIONS: The A630P mutation disrupts the src-homology-2 architecture such that: 1) mutant STAT5b most likely cannot dock to phospho-tyrosines on ligand-activated receptors; and 2) stable interactions with DNA are prevented. Because STAT5b (A630P) is an inefficient signal transducer and transcription factor, the detrimental impact on signaling pathways important for normal growth and immunity explains, in part, the complex clinical phenotype of GH insensitivity and immune dysfunction.

Severe growth hormone insensitivity resulting from total absence of signal transducer and activator of transcription 5b.

CONTEXT: The central clinical feature of GH insensitivity (GHI) is severe growth failure associated with elevated serum concentrations of GH and abnormally low serum levels of IGF-I. GHI can be the result of an abnormality in the GH receptor or aberrancies downstream of the GH receptor. OBJECTIVE: We investigated the GH-IGF-I axis in a young female GHI subject who presented with a height of 114 cm (-7.8 sd score) at age 16.4 yr. PATIENT: The subject, from a consanguineous pedigree, had circulating levels of GH and GH-binding protein that were normal to elevated, whereas IGF-I (7.2 ng/ml; normal, 242-600), IGF-binding protein-3 (543 ng/ml; normal, 2500-4800), and acid-labile subunit (1.22 microg/ml; normal, 5.6-16) levels were abnormally low and failed to increase during an IGF-I generation test. DESIGN: Dermal fibroblast cultures were established with the consent of the patient and family. Immunoblot analysis of cell lysates and DNA sequencing of her signal transducer and activator of transcription 5b (STAT5b), a critical intermediate of the GH-IGF-I axis, were performed. RESULTS: Sequencing of the STAT5b gene revealed a novel homozygous insertion of a single nucleotide in exon 10. The insertion resulted in a frame shift, leading to early protein termination and consequent lack of immunodetectable STAT5b protein. CONCLUSION: The identification of a second case of severe growth failure associated with STAT5b mutation implicates a unique and critical role for STAT5b in GH stimulation of IGF-I gene expression and statural growth.

Transcriptional regulation of insulin-like growth factor-I by interferon-gamma requires STAT-5b.

Insulin-like growth factor (IGF)-I, a growth factor important for cell proliferation, cellular differentiation, and multiple metabolic functions, is regulated in vivo by growth factors and cytokines, but the mechanism(s) of regulation at the cellular level is not well understood. We now demonstrate, employing primary human dermal fibroblasts (CF), that the multipotent cytokine, interferon-gamma (IFN-gamma), can up-regulate IGF-I mRNA expression and that this regulation occurs via activation of the signal transducer and activator of transcription-5b (STAT-5b) pathway. IFN-gamma (100 units/ml) treatment of CF cells resulted in a preferential, time-dependent activation of STAT-5b, although both STAT-5a and STAT-5b isoforms are present. The activated STAT-5b translocated to the nucleus within 30 min of treatment and induced an increase in IGF-I mRNA of 6 +/- 1.0-fold, 3 h post-treatment, with a further increase to 8 +/- 1.7-fold at 5 h. In contrast, IFN-gamma treatment of primary human dermal fibroblasts with a nonfunctional STAT-5b (PF cells) resulted in activation of only STAT-5a and an increase of the IGF-I mRNA level of 1.7 +/- 0.6-fold, 5 h post-treatment. The IFN-gamma-induced regulation of the interferon regulatory factor-1 gene, whose expression is dependent on activated STAT-1, was the same between CF and PF cells. In summary, our results provide evidence of the following in human primary dermal fibroblasts: (a). IFN-gamma preferentially activates STAT-5b, but, in the absence of a functional STAT-5b, STAT-5a is activated; (b). IFN-gamma time-dependently up-regulates IGF-I mRNA expression; (c). the regulation of IGF-I requires an active STAT-5b, and activated STAT-5a cannot substitute for an inactive STAT-5b; and (d). STAT-5b has an essential role in the transcriptional up-regulation of IGF-I.