Pax Transactivation Domain-interacting Protein Research Articles

Pax transactivation domain-interacting protein is required for preserving hematopoietic stem cell quiescence via regulating lysosomal activity.

Hematopoietic stem cells (HSC) maintain lifetime whole blood hematopoiesis through self-renewal and differentiation. In order to sustain HSC stemness, most HSC reside in a quiescence state, which is affected by diverse cellular stress and intracellular signal transduction. How HSC accommodate those challenges to preserve lifetime capacity remains elusive. Here we show that Pax transactivation domain-interacting protein (PTIP) is required for preserving HSC quiescence via regulating lysosomal activity. Using a genetic knockout mouse model to specifically delete Ptip in HSC, we find that loss of Ptip promotes HSC exiting quiescence, and results in functional exhaustion of HSC. Mechanistically, Ptip loss increases lysosomal degradative activity of HSC. Restraining lysosomal activity restores the quiescence and repopulation potency of Ptip-/- HSC. Additionally, PTIP interacts with SMAD2/3 and mediates transforming growth factor-β signaling-induced HSC quiescence. Overall, our work uncovers a key role of PTIP in sustaining HSC quiescence via regulating lysosomal activity.

PTIP Deficiency in B Lymphocytes Ameliorates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice.

Immune dysregulation contributes to the development of ulcerative colitis (UC). The research on the inflammatory response of UC is mainly focused on T cells, with less understanding of the role of B cells. Pax transactivation domain-interacting protein (PTIP) is essential for the development of B cell subpopulations and humoral immunity. The purpose of this study was to elucidate the role of PTIP in B cells of mice with dextran sodium sulfate (DSS)-induced colitis. The B-cell-specific PTIP knockout (PTIP-/-) mice were established by crossbreeding cluster of differentiation (CD)19cre/cre mice with PTIPflox/flox mice. The UC mice were induced by drinking water supplemented with 3.8% Dextran Sulfate Sodium (DSS) (PTIP-/- + DSS). The histological analysis was performed using hematoxylin and eosin staining. The immune cells were isolated using a fluorescence-activated cell sorter. The serum antibodies (immunoglobulin M (IgM) or immunoglobulin G (IgG)) and tumor necrosis factor (TNF)-α were determined by Enzyme linked immunosorbent assay (ELISA). Interestingly, our findings demonstrate that PTIP deficiency in B cells significantly ameliorates UC. In contrast to PTIP-/- + DSS, the wild type (WT) + DSS group showed a more robust increase in disease activity index (DAI) scores (p < 0.05), a substantially shortened colon (p < 0.001) and a decrease of mucous-producing goblet cells and the complete destruction of crypts. Moreover, PTIP-deficient mice manifested markedly altered neutrophil and T-cell distribution in UC (p < 0.05). Although anti-commensal IgG exacerbates UC, we demonstrated, for the first time, that serum natural IgG does not aggravate the pathology of UC. Furthermore, PTIP regulates UC by controlling B-2 cells independently from T cells. Transplantation of splenic B-2 cells from PTIP-deficient mice protected recipient NOD/ShiltJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt (NCG) mice from severe UC.

Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.

Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated Tcells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.

PTIP Deficiency in B Lymphocytes Reduces Subcutaneous Fat Deposition in Mice.

Recent studies have predominantly focused on the role of B cells in metabolic diseases, yet the function of B cells in adipose homeostasis remains unclear. Pax transactivation domain-interacting protein (PTIP), a licensing factor for humoral immunity, is necessary for B cell development and activation. Here, using mice that lack PTIP in B cells (PTIP-/- mice), we explored the role of B cells in adipose homeostasis under physiological conditions. Fat deposition in 8-week-old mice was measured by micro-CT, and PTIP-/- mice presented a marked decrease in the deposition of subcutaneous adipose tissue (SAT). Untargeted lipidomics revealed that the triglyceride composition in SAT was altered in PTIP-/- mice. In addition, there was no difference in the number of adipocyte progenitor cells in the SAT of wild-type (WT) and PTIP-/- mice as measured by flow cytometry. To study the effects of steady-state IgM and IgG antibody levels on fat deposition, PTIP-/- mice were injected intraperitoneally with serum from WT mice once every 3-4 days for 4 weeks. The iSAT mass of the recipient mice showed no significant increase in comparison to the controls after 4 weeks of injections. Our findings reveal that PTIP plays an essential role in regulating subcutaneous adipocyte size, triglyceride composition, and fat deposition under physiological conditions by controlling B cells. The decreased subcutaneous fat deposition in PTIP-/- mice does not appear to be related to the number of adipocyte progenitor cells. The steady-state levels of IgM and IgG antibodies in vivo are not associated with the subcutaneous fat deposition.

The mixed lineage leukemia 4 (MLL4) methyltransferase complex is involved in transforming growth factor beta (TGF-β)-activated gene transcription

ABSTRACTSma and Mad related (SMAD)-mediated Transforming Growth Factor β (TGF-β) and Bone Morphogenetic Protein (BMP) signaling is required for various cellular processes. The activated heterotrimeric SMAD protein complexes associate with nuclear proteins such as the histone acetyltransferases p300, PCAF and the Mixed Lineage Leukemia 4 (MLL4) subunit Pax Transactivation domain-Interacting Protein (PTIP) to regulate gene transcription.We investigated the functional role of PTIP and PTIP Interacting protein 1 (PA1) in relation to TGF-β-activated SMAD signaling. We immunoprecipitated PTIP and PA1 with all SMAD family members to identify the TGF-β and not BMP-specific SMADs as interacting proteins. Gene silencing experiments of MLL4 and the subunits PA1 and PTIP confirm TGF-β-specific genes to be regulated by the MLL4 complex, which links TGF-β signaling to transcription regulation by the MLL4 methyltransferase complex.

PTIP chromatin regulator controls development and activation of B cell subsets to license humoral immunity in mice

B cell receptor signaling and downstream NF-κB activity are crucial for the maturation and functionality of all major B cell subsets, yet the molecular players in these signaling events are not fully understood. Here we use several genetically modified mouse models to demonstrate that expression of the multifunctional BRCT (BRCA1 C-terminal) domain-containing PTIP (Pax transactivation domain-interacting protein) chromatin regulator is controlled by B cell activation and potentiates steady-state and postimmune antibody production in vivo. By examining the effects of PTIP deficiency in mice at various ages during ontogeny, we demonstrate that PTIP promotes bone marrow B cell development as well as the neonatal establishment and subsequent long-term maintenance of self-reactive B-1 B cells. Furthermore, we find that PTIP is required for B cell receptor- and T:B interaction-induced proliferation, differentiation of follicular B cells during germinal center formation, and normal signaling through the classical NF-κB pathway. Together with the previously identified role for PTIP in promoting sterile transcription at the Igh locus, the present results establish PTIP as a licensing factor for humoral immunity that acts at several junctures of B lineage maturation and effector cell differentiation by controlling B cell activation.

The p53-binding protein 1-Tudor-interacting repair regulator complex participates in the DNA damage response

The 53BP1-dependent end-joining pathway plays a critical role in double strand break repair and is uniquely responsible for cellular sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi) in BRCA1-deficient cancers. We and others have investigated the downstream effectors of 53BP1, including replication timing regulatory factor 1 (RIF1) and Pax transactivation domain-interacting protein (PTIP), in the past few years to elucidate how loss of the 53BP1-dependent repair pathway results in PARPi resistance in BRCA1 patients. However, questions regarding the upstream regulation of the 53BP1 pathway remain unanswered. In this study, we identified the Tudor-interacting repair regulator (TIRR) that specifically associates with the ionizing radiation-induced foci formation region of 53BP1. 53BP1 and TIRR form a stable complex, which is required for their expression. Moreover, the 53BP1-TIRR complex dissociates after DNA damage, and this dissociation may be ataxia telangiectasia mutated-dependent. Similar to 53BP1, loss of TIRR restores PARPi resistance in BRCA1-deficient cells. Collectively, our data identified a novel 53BP1-TIRR complex in DNA damage response. TIRR may play both positive and negative roles in 53BP1 regulation. On the one hand, it stabilizes 53BP1 and thus positively regulates 53BP1. On the other hand, its association with 53BP1 prevents 53BP1 localization to sites of DNA damage, and thus TIRR is also an inhibitor of 53BP1.

The role of PAX2 in acute kidney injury and chronic kidney disease

Paired box2(PAX2)is a transciption factor which mainly expressed in the developing kidney.Researches indicate that PAX2 promote the transcription through interactions with the adaptor PAX transactivation domain interacting protein(PTIP). Otherwise, PAX2 protein can lead to chromatin compaction and gene silencing through interactions with Grg4.PAX2 reexpressed in acute kidney injury and involved in promoting cell proliferation.Congenital PAX2 gene mutation is closely related to congenital abnormalies of the kidney and urinary tract.In chronic kidney disease, PAX2 promote proliferation and cyst formation.Here, the recent researches on the function of PAX2 and its role in acute kidney injury and chronic kidney disease are reviewed. Key words: PAX2; Acute kidney injury; Chronic kidney disease

A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

Cell cycle-dependent inhibition of 53BP1 signaling by BRCA1.

DNA damage response mediator protein 53BP1 is a key regulator of non-homologous end-joining (NHEJ) repair. 53BP1 protects DNA broken ends from resection by recruiting two downstream factors, RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), to double-stranded breaks (DSBs) via ATM (ataxia telangiectasia mutated)-mediated 53BP1 phosphorylation, and competes with BRCA1-mediated homologous recombination (HR) repair in G1 phase. In contrast, BRCA1 antagonizes 53BP1-direct NHEJ repair in S/G2 phases. We and others have found that BRCA1 prevents the translocation of RIF1 to DSBs in S/G2 phases; however, the underlying mechanism remains unclear. Here we show that efficient ATM-dependent 53BP1 phosphorylation is restricted to the G1 phase of the cell cycle, as a consequence RIF1 and PTIP accumulation at DSB sites only occur in G1 phase. Mechanistically, both BRCT and RING domains of BRCA1 are required for the inhibition of 53BP1 phosphorylation in S and G2 phases. Thus, our findings reveal how BRCA1 antagonizes 53BP1 signaling to ensure that HR repair is the dominant repair pathway in S/G2 phases.

Uncovering Novel Roles of Tandem BRCA1 C‐terminal (BRCT) Domains in DNA Repair, Genome Stability and Breast Cancer

Pax transactivation domain‐interacting protein (PAXIP1/PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP is primarily a nuclear protein consisting of three pairs of tandem BRCA1 C‐terminal (tBRCT) domains. These BRCT domains are required for its assembly into multiple protein complexes. It is known that mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines and observed differences in protein localization. We have recombinantly expressed the C‐terminal BRCT domains of PTIP, (BRCT36), which retains the ability to form nuclear foci. Using this construct, we set out to investigate how PTIP assembles into DNA repair complexes by conducting differential and ultra‐centrifugation experiments. The biophysical characterization of PTIP uncovers new oligomerization interfaces of higher order structures composed exclusively of BRCT36. These studies indicate that BRCT36 participates in the formation of multiple structurally and stoichiometrically diverse complexes during DNA repair.These studies suggest new and unusual functional roles of PTIP in DNA repair, genome stability and breast cancer.

The Groucho-associated Phosphatase PPM1B Displaces Pax Transactivation Domain Interacting Protein (PTIP) to Switch the Transcription Factor Pax2 from a Transcriptional Activator to a Repressor

Pax genes encode developmental regulatory proteins that specify cell lineages and tissues in metazoans. Upon binding to DNA through the conserved paired domain, Pax proteins can recruit both activating and repressing complexes that imprint distinct patterns of histone methylation associated with either gene activation or silencing. How the switch from Pax-mediated activation to repression is regulated remains poorly understood. In this report, we identify the phosphatase PPM1B as an essential component of the Groucho4 repressor complex that is recruited by Pax2 to chromatin. PPM1B can dephosphorylate the Pax2 activation domain and displace the adaptor protein PTIP, thus inhibiting H3K4 methylation and gene activation. Loss of PPM1B prevents Groucho-mediated gene repression. Thus, PPM1B helps switch Pax2 from a transcriptional activator to a repressor protein. This can have profound implications for developmental regulation by Pax proteins and suggests a model for imprinting specific epigenetic marks depending on the availability of co-factors.

Uncovering novel roles of tandem BRCA1 C‐terminal (BRCT) domains in DNA repair, genome stability and breast cancer (LB236)

Pax transactivation domain‐interacting protein (PTIP) has an important role in preserving genomic stability and is essential for cell survival following DNA damage, although the precise mechanism is undefined. PTIP consists of three pairs of tandem BRCA1 C‐terminal (tBRCT) domains required for assembly into multiple protein complexes. Mutations that disrupt BRCT domain binding correlate with increased cancer rates. To determine its precise role in DNA repair, we profiled the expression of PTIP in breast cancer cell lines. Surprisingly, we’ve discovered a novel truncated isoform of PTIP, containing the last four C‐terminal BRCT domains, (PTIP‐delta). RNAi gene depletion studies reveal a post‐transcriptional origin of PTIP‐delta. Kinetic profiling of PTIP synthesis indicates that a significant portion of PTIP is processed to PTIP‐delta within hours of translation. Recombinant forms of this PTIP‐delta retain the ability to form nuclear repair foci. Biophysical characterization uncovers new oligomerization interfaces of higher order structures composed exclusively of PTIP‐delta. These studies indicate that PTIP‐delta participates in the formation of multiple structurally and stoichiometrically diverse complexes during DNA repair. These studies implicate new and unusual functional roles of PTIP in DNA repair, genome stability and breast cancer.Grant Funding Source: NCI K 22

PITX2 associates with PTIP-containing histone H3 lysine 4 methyltransferase complex

Pituitary homeobox 2 (PITX2), a Paired-like homeodomain transcription factor and a downstream effector of Wnt/β-catenin signaling, plays substantial roles in embryonic development and human disorders. The mechanism of its functions, however, is not fully understood. In this study, we demonstrated that PITX2 associated with histone H3 lysine 4 (H3K4) methyltransferase (HKMT) mixed-lineage leukemia 4 (MLL4/KMT2D), Pax transactivation domain-interacting protein (PTIP), and other H3K4·HKMT core subunits. This association of PITX2 with H3K4·HKMT complex was dependent on PITX2’s homeodomain. Consistently, the PITX2 protein complex was shown to possess H3K4·HKMT activity. Furthermore, the chromatin immunoprecipitation result revealed co-occupancy of PITX2 and PTIP on the promoter of the PITX2’s transcriptional target. Taken together, our data provide new mechanistic perspectives on PITX2’s functions and its related biological processes.

PTIP Associated protein 1, PA1, Is an Independent Prognostic Factor for Lymphnode Negative Breast Cancer

Pax transactivation domain interacting protein (PTIP) associated protein 1, PA1, was a newly found protein participating in the modulation of transactivity of nuclear receptor super family members such as estrogen receptor (ER), androgen receptor (AR) and glucocorticoid receptor (GR). Breast cancer is one of the most life threatening diseases for women and has tight association with estrogen and ER. This study was performed to understand the function of PA1 in breast cancer. The expression of PA1 had been evaluated in a total of 344 primary invasive breast cancer samples and examined the relationship with clinical output, relapse free survival (RFS), breast cancer-specific survival (BCSS). PA1 expression was observed in both nucleus and cytoplasm, however, appeared mainly in nuclear. PA1 nuclear expression was correlated with postmenopausal (P = 0.0097), smaller tumor size (P = 0.0025), negative Ki67 (P = 0.02), positive AR (P = 0.049) and positive ERβ (P = 0.0020). Kaplan–Meier analysis demonstrated PA1 nuclear positive cases seemed to have a longer survival than negative ones for RFS (P = 0.023) but not for BCSS (P = 0.23). In the Cox hazards model, PA1 nuclear protein expression proved to be a significant prognostic univariate parameter for RFS (P = 0.03), but not for BCSS (P = 0.20). In addition, for those patients without lymphnode metastasis PA1 was found to be an independent prognostic factor for RFS (P = 0.025), which was verified by univariate and multivariate analyses. These investigations suggested PA1 expression could be a potential prognostic indicator for RFS in breast cancer.

Mechanisms of gene activation and repression by Pax proteins in the developing kidney

During embryonic development, DNA binding proteins help specify and restrict the fates of pluripotent stem cells. In the developing kidney, Pax2 proteins are among the earliest markers for the renal epithelial cell lineage, with expression in the mesenchyme and in proliferating epithelia. The Pax2 protein is essential for interpreting inductive signals emanating from the ureteric bud such that the kidney mesenchyme can convert to epithelia. The biochemistry of Pax protein function is being studied in a variety of model systems. Through interactions with the adaptor Pax transactivation-domain interacting protein (PTIP), Pax proteins can recruit members of the Trithorax family of histone methyltransferases to imprint activating epigenetic marks on chromatin. However, interactions with the corepressor Groucho-related gene-4 (Grg4) protein can inhibit activation and instead recruit Polycomb repressor complexes to promote target-gene silencing. We present a model whereby the regulated interactions of Pax proteins with available cofactor-mediated activation or gene silencing at different stages of development. The implications for establishing and maintaining the epigenome are discussed.

8: Epigenetic regulation of Th-17 differentiation

Differentiation of Th17 cells requires the combination of IL-6 and TGF-β and is further maintained by IL-23. This T cell subtype is important for both host defense against extracellular pathogens and the pathogenesis of autoimmune diseases. The main cytokines produced by Th17 cells are IL-17, which induces strong proinflammatory effects, and IL-22, an enhancer of epithelial proliferation and healing responses. Previous research has demonstrated that the expression of the transcription factor c-Maf is important for inhibition of IL-22 production in Th17 cells. The transcription factor AHR (aryl hydrocarbon receptor) has also been shown crucial for proper Th17 differentiation. The adapter protein PTIP (Pax transactivation domain-interacting protein) has recently been shown to have a role in the maturation of thymocytes. PTIP stably associates with complexes containing the chromatin methyltransferases MLL3 and MLL4, which modify chromatin in order to activate gene transcription. Here we demonstrate that PTIP contributes to Th17 differentiation and function via regulation of both c-Maf and AHR. Using CD4+ T cells from Ptipf/fCD4 Cre+ (PTIP−/−) mice and Cre- (WT) controls in a T cell transfer model of chronic colitis, we determined that RAG2−/− mice receiving WT cells had aggressive colitis with high expression of IL-17 in the intestine. However, in mice receiving PTIP−/− cells there was a reduction in IL-17 and an increase in IL-22 expression. The change in IL-17 and IL-22 expression in PTIP−/− mice was confirmed in vitro. Further analysis by chromatin immunoprecipitation (ChIP) assay showed that MLL3 binding and its dependent chromatin modifications at the loci for IL-17, c-Maf and AHR were enhanced in WT Th17 cells when compared to Th0 conditions and reduced in PTIP−/− Th17 cells. These data highlight, for the first time, that PTIP is essential for proper differentiation of Th17 cells.

A polyglutamine expansion disease protein sequesters PTIP to attenuate DNA repair and increase genomic instability

Glutamine (Q) expansion diseases are a family of degenerative disorders caused by the lengthening of CAG triplet repeats present in the coding sequences of seemingly unrelated genes whose mutant proteins drive pathogenesis. Despite all the molecular evidence for the genetic basis of these diseases, how mutant poly-Q proteins promote cell death and drive pathogenesis remains controversial. In this report, we show a specific interaction between the mutant androgen receptor (AR), a protein associated with spinal and bulbar muscular atrophy (SBMA), and the nuclear protein PTIP (Pax Transactivation-domain Interacting Protein), a protein with an unusually long Q-rich domain that functions in DNA repair. Upon exposure to ionizing radiation, PTIP localizes to nuclear foci that are sites of DNA damage and repair. However, the expression of poly-Q AR sequesters PTIP away from radiation-induced nuclear foci. This results in sensitivity to DNA-damaging agents and chromosomal instabilities. In a mouse model of SBMA, evidence for DNA damage is detected in muscle cell nuclei and muscular atrophy is accelerated when one copy of the gene encoding PTIP is removed. These data provide a new paradigm for understanding the mechanisms of cellular degeneration observed in poly-Q expansion diseases.

Identification and Characterization of a Novel Nuclear Protein Complex Involved in Nuclear Hormone Receptor-mediated Gene Regulation

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of approximately 1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-alpha was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.

Pax Transactivation-Domain Interacting Protein Is Required for Urine Concentration and Osmotolerance in Collecting Duct Epithelia

Pax transactivation-domain interacting protein (PTIP) is a widely expressed nuclear protein that is essential for early embryonic development. PTIP was first identified on the basis of its interactions with the developmental regulator Pax2 but can also bind to other nuclear transcription factors. The Pax2 protein is essential for development of the renal epithelia and for regulating the response of mature collecting ducts to hyperosmotic stress. For determination of whether PTIP also functions in more differentiated cell types, the Cre-LoxP system was used to delete the ptip gene in the renal collecting ducts using Ksp-Cre driver mice. Collecting duct-specific ptip knockout mice were viable with little discernible phenotype under normal physiologic conditions. However, collecting duct-specific ptip mutants were unable to concentrate urine after the treatment of desamino-cis, D-arginine vasopressin, an antidiuretic hormone. Furthermore, aquaporin-2 (AQP2) expression in the inner medulla of the ptip knockout mice was decreased approximately 10-fold compared with that of wild-type littermates. Expression level of tonicity responsive enhancer binding protein, a transcription factor of AQP2, is not altered in the mutant mice, but its nuclear localization in the inner medulla is unresponsive after treatment with vasopressin agonists. This was due, at least in part, to decreased expression of the arginine vasopressin receptor 2 in ptip mutants. Furthermore, ptip null inner medullary collecting duct cells were sensitive to hyperosmolality in vitro. Thus, ptip is required for the urine concentration mechanism by modulating arginine vasopressin receptor 2 and AQP2 expression in the inner medulla. The data suggest an essential role for ptip in regulating urine concentration and in controlling survival of collecting duct epithelial cells in high osmolality.