Investigating Protein-Protein Interactions of Autophagy-Involved TNIP1.

Myriad proteins are involved in the process of autophagy, which they participate in via their protein-protein interactions (PPI). Herein we outline a methodology for examining such interactions utilizing the case of intrinsically disordered protein (IDP) TNIP1 and its interaction with linear M1-linked polyubiquitin. This includes methods for recombinant production, purification, immuno-identification, and analysis of an IDP associated with autophagy, its ordered binding partner, and means of quantitatively analyzing their interaction.

Repressive Control of Keratinocyte Cytoplasmic Inflammatory Signaling.

The overactivity of keratinocyte cytoplasmic signaling contributes to several cutaneous inflammatory and immune pathologies. An important emerging complement to proteins responsible for this overactivity is signal repression brought about by several proteins and protein complexes with the native role of limiting inflammation. The signaling repression by these proteins distinguishes them from transmembrane receptors, kinases, and inflammasomes, which drive inflammation. For these proteins, defects or deficiencies, whether naturally arising or in experimentally engineered skin inflammation models, have clearly linked them to maintaining keratinocytes in a non-activated state or returning cells to a post-inflamed state after a signaling event. Thus, together, these proteins help to resolve acute inflammatory responses or limit the development of chronic cutaneous inflammatory disease. We present here an integrated set of demonstrated or potentially inflammation-repressive proteins or protein complexes (linear ubiquitin chain assembly complex [LUBAC], cylindromatosis lysine 63 deubiquitinase [CYLD], tumor necrosis factor alpha-induced protein 3-interacting protein 1 [TNIP1], A20, and OTULIN) for a comprehensive view of cytoplasmic signaling highlighting protein players repressing inflammation as the needed counterpoints to signal activators and amplifiers. Ebb and flow of players on both sides of this inflammation equation would be of physiological advantage to allow acute response to damage or pathogens and yet guard against chronic inflammatory disease. Further investigation of the players responsible for repressing cytoplasmic signaling would be foundational to developing new chemical-entity pharmacologics to stabilize or enhance their function when clinical intervention is needed to restore balance.

Thermostable Proteins from HaCaT Keratinocytes Identify a Wide Breadth of Intrinsically Disordered Proteins and Candidates for Liquid-Liquid Phase Separation.

Intrinsically disordered proteins (IDPs) move through an ensemble of conformations which allows multitudinous roles within a cell. Keratinocytes, the predominant cell type in mammalian epidermis, have had only a few individual proteins assessed for intrinsic disorder and its possible contribution to liquid-liquid phase separation (LLPS), especially in regard to what functions or structures these proteins provide. We took a holistic approach to keratinocyte IDPs starting with enrichment via the isolation of thermostable proteins. The keratinocyte protein involucrin, known for its resistance to heat denaturation, served as a marker. It and other thermostable proteins were identified by liquid chromatography tandem mass spectrometry and subjected to extensive bioinformatic analysis covering gene ontology, intrinsic disorder, and potential for LLPS. Numerous proteins unique to keratinocytes and other proteins with shared expression in multiple cell types were identified to have IDP traits (e.g., compositional bias, nucleic acid binding, and repeat motifs). Among keratinocyte-specific proteins, many that co-assemble with involucrin into the cell-specific structure known as the cornified envelope scored highly for intrinsic disorder and potential for LLPS. This suggests intrinsic disorder and LLPS are previously unrecognized traits for assembly of the cornified envelope, echoing the contribution of intrinsic disorder and LLPS to more widely encountered features such as stress granules and PML bodies.

Intrinsic disorder in integral membrane proteins.

The well-defined roles and specific protein-protein interactions of many integral membrane proteins (IMPs), such as those functioning as receptors for extracellular matrix proteins and soluble growth factors, easily align with considering IMP structure as a classical "lock-and-key" concept. Nevertheless, continued advances in understanding protein conformation, such as those which established the widespread existence of intrinsically disordered proteins (IDPs) and especially intrinsically disordered regions (IDRs) in otherwise three-dimensionally organized proteins, call for ongoing reevaluation of transmembrane proteins. Here, we present basic traits of IDPs and IDRs, and, for some select single-span IMPs, consider the potential functional advantages intrinsic disorder might provide and the possible conformational impact of disease-associated mutations. For transmembrane proteins in general, we highlight several investigational approaches, such as biophysical and computational methods, stressing the importance of integrating them to produce a more-complete mechanistic model of disorder-containing IMPs. These procedures, when synergized with in-cell assessments, will likely be key in translating in silico and in vitro results to improved understanding of IMP conformational flexibility in normal cell physiology as well as disease, and will help to extend their potential as therapeutic targets.

Seeing Keratinocyte Proteins through the Looking Glass of Intrinsic Disorder.

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid-liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.

The Anti-Inflammatory Protein TNIP1 Is Intrinsically Disordered with Structural Flexibility Contributed by Its AHD1-UBAN Domain.

TNFAIP3 interacting protein 1 (TNIP1) interacts with numerous non-related cellular, viral, and bacterial proteins. TNIP1 is also linked with multiple chronic inflammatory disorders on the gene and protein levels, through numerous single-nucleotide polymorphisms and reduced protein amounts. Despite the importance of TNIP1 function, there is limited investigation as to how its conformation may impact its apparent multiple roles. Hub proteins like TNIP1 are often intrinsically disordered proteins. Our initial in silico assessments suggested TNIP1 is natively unstructured, featuring numerous potentials intrinsically disordered regions, including the ABIN homology domain 1-ubiquitin binding domain in ABIN proteins and NEMO (AHD1-UBAN) domain associated with its anti-inflammatory function. Using multiple biophysical approaches, we demonstrate the structural flexibility of full-length TNIP1 and the AHD1-UBAN domain. We present evidence the AHD1-UBAN domain exists primarily as a pre-molten globule with limited secondary structure in solution. Data presented here suggest the previously described coiled-coil conformation of the crystallized UBAN-only region may represent just one of possibly multiple states for the AHD1-UBAN domain in solution. These data also characterize the AHD1-UBAN domain in solution as mostly monomeric with potential to undergo oligomerization under specific environmental conditions (e.g., binding partner availability, pH-dependence). This proposed intrinsic disorder across TNIP1 and within the AHD1-UBAN region is likely to impact TNIP1 function and interaction with its multiple partners.

Enhanced Wound Healing- and Inflammasome-Associated Gene Expression in TNFAIP3-Interacting Protein 1- (TNIP1-) Deficient HaCaT Keratinocytes Parallels Reduced Reepithelialization.

TNIP1 protein is a widely expressed, cytoplasmic inhibitor of inflammatory signaling initiated by membrane receptors such as TLRs which recognize pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs). Keratinocyte TNIP1 deficiency sensitizes cells to PAMPs and DAMPs promoting hyperresponsive expression and secretion of cytokine markers (e.g., IL-8 and IL-6) relevant to cases of chronic inflammation, like psoriasis, where TNIP1 deficiency has been reported. Here, we examined the impact of TNIP1 deficiency on gene expression and cellular responses (migration and viability) relevant to acute inflammation as typically occurs in wound healing. Using siRNA-mediated TNIP1 expression knockdown in cultured HaCaT keratinocytes, we investigated TNIP1 deficiency effects on signaling downstream of TLR3 agonism with low-concentration poly (I:C), a representative PAMP/DAMP. The combination of TNIP1 knockdown and PAMP/DAMP signaling disrupted expression of specific keratinocyte differentiation markers (e.g., transglutaminase 1 and involucrin). These same conditions promoted synergistically increased expression of wound-associated markers (e.g., S100A8, TGFß, and CCN2) suggesting potential benefit of increased inflammatory response from reduced TNIP1 protein. Unexpectedly, poly (I:C) challenge of TNIP1-deficient cells restricted reepithelialization and reduced cell viability. In these cells, there was not only increased expression for genes associated with inflammasome assembly (e.g., ASC, procaspase 1) but also for A20, a TNIP1 partner protein that represses cell-death signaling. Despite this possibly compensatory increase in A20 mRNA, there was a decrease in phospho-A20 protein, the form necessary for quenching inflammation. Hyperresponsiveness to poly (I:C) in TNIP1-deficient keratinocytes was in part mediated through p38 and JNK pathways. Taken together, we conclude that TNIP1 deficiency promotes enhanced expression of factors associated with promoting wound healing. However, the coupled, increased potential priming of the inflammasome and reduced compensatory activity of A20 has a net negative effect on overall cell recovery potential manifested by poor reepithelialization and viability. These findings suggest a previously unrecognized role for TNIP1 protein in limiting inflammation during successful progression through early wound healing stages.

In Silico and In Vitro Considerations of Keratinocyte Nuclear Receptor Protein Structural Order for Improving Experimental Analysis.

Nuclear receptors (NR) regulate gene expression critical in keratinocyte replication and differentiation. In addition to a ligand-binding domain, NR like other transcription factor families have a DNA-binding domain that must attain a particular conformation for effective interaction with the three-dimensional structure in promoters of target genes for control of their expression. Such protein-DNA assemblies extend the classic "lock and key" idea typified by protein-protein interactions. However, it is becoming increasingly clear that multi-subdomain transcription factors like NR frequently range along the length of the protein from structured, ordered regions expected for interaction with a preset partner to more flexible, intrinsically disordered regions which are more available for diverse posttranslational modifications and/or interaction with differing partners. The extended amino terminus of NR (the A/B subdomain) is one such intrinsically disordered region. Here we provide a primer on in silico-based recognition of amino acid composition and order associated with such conformational flexibility along with adaptations of readily accessible laboratory techniques (e.g., considerations for recombinant expression, sensitivity to protease and proteasome digestion) to facilitate initial prediction and testing for intrinsic disorder in various proteins of interest to keratinocyte biologists, like NR and other transcription factors.

TNIP1 in Autoimmune Diseases: Regulation of Toll-like Receptor Signaling.

TNIP1 protein is increasingly being recognized as a key repressor of inflammatory signaling and a potential factor in multiple autoimmune diseases. In addition to earlier foundational reports of TNIP1 SNPs in human autoimmune diseases and TNIP1 protein-protein interaction with receptor regulating proteins, more recent studies have identified new potential interaction partners and signaling pathways likely modulated by TNIP1. Subdomains within the TNIP1 protein as well as how they interact with ubiquitin have not only been mapped but inflammatory cell- and tissue-specific consequences subsequent to their defective function are being recognized and related to human disease states such as lupus, scleroderma, and psoriasis. In this review, we emphasize receptor signaling complexes and regulation of cytoplasmic signaling steps downstream of TLR given their association with some of the same autoimmune diseases where TNIP1 has been implicated. TNIP1 dysfunction or deficiency may predispose healthy cells to the inflammatory response to otherwise innocuous TLR ligand exposure. The recognition of the anti-inflammatory roles of TNIP1 and improved integrated understanding of its physical and functional association with other signaling pathway proteins may position TNIP1 as a candidate target for the design and/or testing of next-generation anti-inflammatory therapeutics.

TNIP1 reduction sensitizes keratinocytes to post-receptor signalling following exposure to TLR agonists.

Cell level inflammatory signalling is a combination of initiation at cell membrane receptors and modulation by cytoplasmic regulatory proteins. For keratinocytes, the predominant cell type in the epidermis, this would include toll-like receptors (TLR) and cytoplasmic proteins that propagate or dampen post-receptor signalling. We previously reported that increased levels of tumor necrosis factor α induced protein 3-interacting protein 1 (TNIP1) in HaCaT keratinocytes leads to decreased expression of stress response and inflammation-associated genes. This finding suggested decreased TNIP1 levels, as seen in some cutaneous disease states, may produce the opposite effect, sensitizing cells to triggers of inflammatory signalling including those sensed by TLR. In this study of TNIP1-deficient HaCaT keratinocytes we examined intracellular signalling consequences especially those expected to produce gene expression changes downstream of TLR3 or TLR2/6 activation by Poly (I:C) or FSL-1, agonists modeling skin relevant pathogens. We found TNIP1-deficient keratinocytes are hyper-sensitive to TLR activation compared to control cells with a normal complement of TNIP1 and receiving the same agonist stimulation. TNIP1-deficient keratinocytes have increased levels of activated (phosphorylated) cytoplasmic mediators such as JNK and p38 and greater nuclear translocation of NF-κB and phospho-p38 when exposed to TLR ligands. This is consistent with significantly increased expression of several inflammatory cytokines and chemokines, such as IL-6 and IL-8. These results describe how decreased TNIP1 levels promote a hyper-sensitive state in HaCaT keratinocytes evidenced by increased activation of signalling molecules downstream of TLR agonists and increased expression of pro-inflammatory mediators. TNIP1 keratinocyte deficiency as reported for some skin diseases may predispose these cells to excessive inflammatory signalling upon exposure to viral or bacterial ligands for TLR.

Interleukin-17 signaling triggers degradation of the constitutive NF-κB inhibitor ABIN-1.

IL-17 activates NF-κB and inducing expression of proinflammatory genes. IL-17 drives disease in autoimmune conditions, and anti-IL-17 antibodies have shown impressive success in the clinic. Although produced by lymphocytes, IL-17 predominantly signals in fibroblasts and epithelial cells. IL-17-driven inflammation is kept in check by negative feedback signaling molecules, including the ubiquitin editing enzyme A20, whose gene TNFΑIP3 is and similarly linked to autoimmune disease susceptibility. Accordingly, we hypothesized that ABIN-1 might play a role in negatively regulating IL-17 signaling activity. Indeed, ABIN-1 enhanced both tonic and IL-17-dependent NF-κB signaling in IL-17-responsive fibroblast cells. Interestingly, the inhibitory activities of ABIN-1 on IL-17 signaling were independent of A20. ABIN-1 is a known NF-κB target gene, and we found that IL-17-induced activation of NF-κB led to enhanced ABIN-1 mRNA expression and promoter activity. Surprisingly, however, the ABIN-1 protein was inducibly degraded following IL-17 signaling in a proteasome-dependent manner. Thus, ABIN-1, acting independently of A20, restricts both baseline and IL-17-induced inflammatory gene expression. We conclude that IL-17-induced signals lead to degradation of ABIN-1, thereby releasing a constitutive cellular brake on NF-κB activation.

Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions.

Epidermal keratinocytes serve as the primary barrier between the body and environmental stressors. They are subjected to numerous stress events and are likely to respond with a repertoire of heat shock proteins (HSPs). HSPA6 (HSP70B') is described in other cell types with characteristically low to undetectable basal expression, but is highly stress induced. Despite this response in other cells, little is known about its control in keratinocytes. We examined endogenous human keratinocyte HSPA6 expression and localized some responsible transcription factor sites in a cloned HSPA6 3 kb promoter. Using promoter 5' truncations and deletions, negative and positive regulatory regions were found throughout the 3 kb promoter. A region between -346 and -217 bp was found to be crucial to HSPA6 basal expression and stress inducibility. Site-specific mutations and DNA-binding studies show that a previously uncharacterized AP1 site contributes to the basal expression and maximal stress induction of HSPA6. Additionally, a new heat shock element (HSE) within this region was defined. While this element mediates increased transcriptional response in thermally stressed HaCaT keratinocytes, it preferentially binds a stress-inducible factor other than heat shock factor (HSF)1 or HSF2. Intriguingly, this newly characterized HSPA6 HSE competes HSF1 binding a consensus HSE and binds both HSF1 and HSF2 from other epithelial cells. Taken together, our results demonstrate that the HSPA6 promoter contains essential negative and positive promoter regions and newly identified transcription factor targets, which are key to the basal and stress-inducible expression of HSPA6. Furthermore, these results suggest that an HSF-like factor may preferentially bind this newly identified HSPA6 HSE in HaCaT cells.

TNIP1 reduction of HSPA6 gene expression occurs in promoter regions lacking binding sites for known TNIP1-repressed transcription factors.

TNFα-induced protein 3-interacting protein 1 (TNIP1) represses signaling pathways initiated by specific nuclear and transmembrane receptors. This effect results in reduced activity of distinct transcription factors such as retinoic acid receptors (RAR), peroxisome-proliferator-activated receptors (PPAR), and NFκB. TNIP1-null and TNIP1-knockin defective for ubiquitin-binding mice show increased liver apoptosis, and enlarged spleen and lymph nodes, respectively. To complement current knowledge of TNIP1's broad physiologic functions as interpreted from in vivo studies and specific expression consequences from transcription factor repression, we determined effects of excess TNIP1 on global gene regulation. Following experimentally increased expression of TNIP1 in cultured keratinocytes, our gene expression microarray analysis not only confirmed TNIP1's association in previously known pathways and functions but also found a novel TNIP1-regulated pathway - the cell stress response. Under standard culture conditions, expression of several heat shock proteins, including HSPA1A, HSPA6, DNAJA1 and DNAJB1, was reduced. In heat-stressed conditions, differential regulation of HSPA1A and HSPA6 was observed, where only HSPA6 expression was reduced after heat-shock. Using HSPA6 as a model to elucidate the mechanism of the TNIP1-mediated HSP repression, we determined that TNIP1 likely represses HSPs through factors other than RAR, PPAR or NFκB despite the presence of these factors' binding sites in the HSPA6 promoter. These results indicate that regulation of HSPs may be through a yet unknown TNIP1-associated pathway. Additionally, these results suggest that TNIP1's reduction of HSP expression levels could negatively impact HSP chaperone capacity or their participation in the cell stress response.

Transgene delivery to cultured keratinocytes via replication-deficient adenovirus vectors.

Transient transgene expression can facilitate investigation of that gene-product function or effect on keratinocyte biology. Several chemical and biologic delivery systems are available, and among them adenoviruses offer particular advantages in efficiency and transgene capacity. Here we describe the advantages of bicistronic adenovirus and inclusion of the polycation hexadimethrine bromide to aid in the detection of positively transduced cells and enhance transduction efficiency.

Sp sites contribute to basal and inducible expression of the human TNIP1 (TNFα-inducible protein 3-interacting protein 1) promoter.

TNIP1 [TNFα (tumour necrosis factor α)-induced protein 3-interacting protein 1] is a co-repressor of RAR (retinoic acid receptor) and PPAR (peroxisome-proliferator-activated receptor). Additionally, it can reduce signalling stemming from cell membrane receptors such as those for TNFα and EGF (epidermal growth factor). Consequently, it influences a variety of receptor-mediated events as diverse as transcription, programmed cell death and cell cycling. Thus changes in TNIP1 expression levels are likely to affect multiple important biological end points. TNIP1 expression level changes have been linked to psoriasis and systemic sclerosis. As such, it is crucial to determine what controls its expression levels, starting with constitutive control of its promoter. Our analysis of the TNIP1 promoter revealed multiple transcription start sites in its GC-rich proximal regions along with two transcriptionally active Sp (specificity protein) sites, responsive to both Sp1 and Sp3. EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) demonstrated physical binding between Sp1 and Sp3 at these sites. A decrease in Sp1 protein levels via siRNA (short interfering RNA) or diminished Sp1 DNA binding by mithramycin decreased TNIP1 mRNA levels. This Sp-binding GC-rich region of the TNIP1 promoter also participates in transcriptional activation by ligand-bound RAR. Together, these results demonstrate newly identified regulators of TNIP1 expression and suggest possible transcription factor targets which in turn control TNIP1-related biological end points ranging from apoptosis to inflammatory diseases.

Human TNFα-induced protein 3-interacting protein 1 (TNIP1) promoter activation is regulated by retinoic acid receptors.

Coregulator proteins play key roles in transcriptional control by members of the nuclear receptor superfamily. Previously, we demonstrated that tumor necrosis factor α (TNFα)-induced protein 3-interacting protein 1 (TNIP1) is a corepressor of agonist-bound retinoic acid receptors (RARs). Additionally, TNIP1 has been shown to repress peroxisome proliferator-activated receptors (PPAR) and NF-κB activity and interact with HIV proteins nef and matrix. TNIP1 transcriptional regulation, however, is under studied. Here we show that under permissive epigenetic conditions, TNIP1 expression is induced by all trans retinoic acid (ATRA). Within a 6000 bp region of the human TNIP1 promoter we cloned, both proximal and distal promoter regions are RAR responsive with the latter having RA response elements (RAREs) recognizable by their sequence and functionality in native promoter and synthetic RARE luciferase constructs, EMSA, and ChIP assays. These findings suggest a feedback loop whereby RARs activate expression of TNIP1, which then attenuates their activity. Together with anticipated constitutive transcription factors and the previously described NF-κB-responsiveness of the proximal TNIP1 promoter, the expected combinatorial control of TNIP1 expression could likely modulate TNIP1's impact in any of its target pathways. The degree of control by RARs or other transcription factors would in turn depend on their cell-specific level of expression and/or activation from signals in the environment such as ATRA and TNFα.

Organotypic modeling of human keratinocyte response to peroxisome proliferators.

Peroxisome proliferators (PPs) are a diverse chemical group including hypolipidemic drugs and some fatty acids. Their stimulation of PP-activated receptors (PPARs) and subsequent control of gene expression regulates metabolism and differentiation in many cells. PPs have multiple opportunities to target human epidermal keratinocytes because of delivery through dietary, clinical, and/or topical exposure routes. PPAR knockout mice and PP treatment of mouse skin or human keratinocytes in monolayer culture have established some effects for PPs in cutaneous differentiation. However, incomplete epidermal maturation characteristic of monolayer keratinocytes and rodent-specific effects may limit our full understanding of human keratinocyte responses to PPs. To address these issues, we investigated PP influence on primary human keratinocytes in organotypic cultures that recapitulate biochemical markers of epidermis. We found that the PPARα agonists clofibrate, docasohexaenoic acid, and WY-14,643 produced mild to moderate keratinocyte hyperplasia, increased stratification (particularly of granular and cornified layers), and enhanced levels of the differentiation markers filaggrin, ABCA12, and phosphorylated HSP27. Several PP effects generated in the organotypic system, however, were distinct from those previously reported for rodent skin and human keratinocyte monolayer cultures, suggesting that the species and growth context of target cells can impact exposure outcomes. Given the utility of organotypic cultures for modeling the epidermis, studies in this system may bridge the gap between the rodent assays and clinical studies of human epidermal responses to PPs.

Emerging roles for TNIP1 in regulating post-receptor signaling.

A vast number of cellular processes and signaling pathways are regulated by various receptors, ranging from transmembrane to nuclear receptors. These receptor-mediated processes are modulated by a diverse set of regulatory proteins. TNFα-induced protein 3-interacting protein 1 is such a protein that inhibits both transduction by transmembrane receptors, such as TNFα-receptor, EGF-R, and TLR, and nuclear receptors' PPAR and RAR activity. These receptors play key roles in regulating inflammation and inflammatory diseases. A growing number of references have implicated TNIP1 through GWAS and expression studies in chronic inflammatory diseases such as psoriasis and rheumatoid arthritis, although TNIP1s exact role has yet been determined. In this review, we aim to integrate the current knowledge of TNIP1s functions with the diseases in which it has been associated to potentially elucidate the role this regulator has in promoting or alleviating these inflammatory diseases.

PPARγ and NF-κB regulate the gene promoter activity of their shared repressor, TNIP1.

Human TNFAIP3 interacting protein 1 (TNIP1) has diverse functions including support of HIV replication through its interaction with viral Nef and matrix proteins, reduction of TNFα-induced signaling through its interaction with NF-κB pathway proteins, and corepression of agonist-bound retinoic acid receptors and peroxisome proliferator-activated receptors (PPAR). The wide tissue distribution of TNIP1 provides the opportunity to influence numerous cellular responses in these roles and defining control of TNIP1 expression would be central to improved understanding of its impact on cell function. We cloned 6kb of the human TNIP1 promoter and performed predictive and functional analyses to identify regulatory elements. The promoter region proximal to the transcription start site is GC-rich without a recognizable TATA box. In contrast to this proximal ~500bp region, 6kb of the promoter increased reporter construct constitutive activity over five-fold. Throughout the 6kb length, in silico analysis identified several potential binding sites for both constitutive and inducible transcription factors; among the latter were candidate NF-κB binding sequences and peroxisome proliferator response elements (PPREs). We tested NF-κB and PPAR regulation of the endogenous TNIP1 gene and cloned promoter by expression studies, electrophoretic mobility shift assays, and chromatin immunoprecipitations. We validated NF-κB sites in the TNIP1 promoter proximal and distal regions as well as one PPRE in the distal region. The ultimate control of the TNIP1 promoter is likely to be a combination of constitutive transcription factors and those subject to activation such as NF-κB and PPAR.

TNIP1, a retinoic acid receptor corepressor and A20-binding inhibitor of NF-κB, distributes to both nuclear and cytoplasmic locations.

An increasingly wide range of functions, from repression of NF-κB signaling to protection from apoptosis, is being recognized for tumor necrosis factor α-induced protein 3-interacting protein 1 (TNIP1). The authors recently demonstrated TNIP1 interaction with and repression of liganded retinoic acid receptors, distinguishing it from the more typical NCoR and SMRT corepressors, which function only in the absence of ligand. To improve their understanding of TNIP1's roles in physiologic and pathologic events, the authors examined its distribution in normal and malignant human tissues and cultured cells. They found cytoplasmic and nuclear TNIP1 in normal skin keratinocytes as it colocalized with retinoic acid receptor α, one of the nuclear receptors it corepresses. Nuclear and cytoplasmic TNIP1 was also found in the malignant keratinocytes of squamous cell carcinomas. Compared to adjacent normal tissues of other organs, TNIP1 staining and distribution varied with increased levels in esophageal cancer and marked decreases in prostate cancer. The varying levels and distribution of TNIP1 in normal and disease state tissues could be expected to affect processes in which TNIP1 is involved, such as NF-κB and nuclear receptor signaling, possibly contributing to the disease course or response to therapies targeting these key players of cell growth and differentiation.