Pontin is required for pre-TCR signaling at the β-selection checkpoint in T cell development

Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Based on high expression in lymphoid tissues, we examined whether Pontin has a T cell-specific function. We generated Pontin^f/f;Lck-Cre mice, in which Pontin can be conditionally deleted in T cells and then explored T cell-specific function of Pontin in vivo. Here, we show that specific abrogation of Pontin expression in T cells almost completely blocked development of αβ T cells at the β-selection checkpoint by inducing cell apoptosis indicating that Pontin is essential for early T cell development. Pontin-deficient thymocytes show a comparable expression level of T cell receptor (TCR)β chain, but have enhanced activation of p53 and Notch signaling compared to wild-type thymocytes. Intriguingly, the developmental block of αβ T cells can be partially rescued by loss of p53. Together, our data demonstrate a novel role of Pontin as a crucial regulator in pre-TCR signaling during T cell development.     

Notch3 is dispensable for thymocyte β-selection and Notch1-induced T cell leukemogenesis

Notch1 (N1) signaling induced by intrathymic Delta-like (DL) ligands is required for T cell lineage commitment as well as self-renewal during “β-selection” of TCRβ⁺ CD4⁻CD8⁻ double negative 3 (DN3) T cell progenitors. However, over-expression of the N1 intracellular domain (ICN1) renders N1 activation ligand-independent and drives leukemic transformation during β-selection. DN3 progenitors also express Notch3 (N3) mRNA, and over-expression of ligand-independent mutant N3 (ICN3) influences β-selection and drives T cell leukemogenesis. However, the importance of ligand-activated N3 in promoting β-selection and ICN1-induced T cell leukemogenesis has not been examined. To address these questions we generated mice lacking functional N3. We confirmed that DN3 progenitors express N3 protein using a N3-specific antibody. Surprisingly however, N3-deficient DN3 thymocytes were not defective in generating DP thymocytes under steady state conditions or in more stringent competition assays. To determine if N3 co-operates with N1 to regulate β-selection, we generated N1;N3 compound mutants. However, N3 deficiency did not exacerbate the competitive defect of N1^+/− DN3 progenitors, demonstrating that N3 does not compensate for limiting N1 during T cell development. Finally, N3 deficiency did not attenuate T cell leukemogenesis induced by conditional expression of ICN1 in DN3 thymocytes. Importantly, we showed that in contrast to N1, N3 has a low binding affinity for DL4, the most abundant intrathymic DL ligand. Thus, despite the profound effects of ectopic ligand-independent N3 activation on T cell development and leukemogenesis, physiologically activated N3 is dispensable for both processes, likely because N3 interacts poorly with intrathymic DL4.     

LPA-producing enzyme PA-PLA₁α regulates hair follicle development by modulating EGFR signalling

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.     

KDM6B regulates M2 polarization of macrophages by modulating the stability of nuclear β-catenin

Accumulating evidences suggest that the epigenetic regulation plays a pivotal role in establishing phenotype and function of tumor associated macrophages (TAMs). KDM6B is an epigenetic enzyme responsible for the H3K27me3 and reported to influence macrophage polarization. However, the underlying mechanism remains to be determined. Here, we demonstrated that inhibition of KDM6B in TAMs increased M2 polarization induced by coculture of breast cancer cells. Furthermore, we identified that KDM6B downregulation activated β-catenin/c-Myc signaling, and thus promoted the M2-like phenotype. KDM6B accelerated the intranuclear ubiquitination degradation of β-catenin, which depended on its demethylase activity. Therapeutically, our data showed that activated vitamin D analog paricalcitol upregulated the expression of KDM6B and decreased the M2 polarization, consequently protected against tumor progress in the xenograft mouse model of breast cancer. Taken together, our data reveal that epigenetic regulator KDM6B prevents M2 polarization via promoting the intranuclear degradation of β-catenin. Active vitamin D analog induces KDM6B and suppresses tumor progress, suggesting a novel therapeutic potential of epigenetic modulation for the tumor treatment.     

Wnt/β-catenin signaling in the dental mesenchyme regulates incisor development by regulating Bmp4

Loss- and gain-of function approaches modulating canonical Wnt/β-catenin activity have established a role for the Wnt/β-catenin pathway during tooth development. Here we show that Wnt/β-catenin signaling is required in the dental mesenchyme for normal incisor development, as locally restricted genetic inactivation of β-catenin results in a splitting of the incisor placode, giving rise to two incisors. Molecularly this is first associated with down-regulation of Bmp4 and subsequent splitting of the Shh domain at a subsequent stage. The latter phenotype can be mimicked by ectopic application of the BMP antagonist Noggin. Conditional genetic inactivation of Bmp4 in the mesenchyme reveals that mesenchymal BMP4 activity is required for maintenance of Shh expression in the dental ectoderm. Taken together our results indicate that β-catenin together with Lef1 and Tcf1 are required to activate Bmp4 expression in order to maintain Shh expression in the dental ectoderm. This provides a mechanism whereby the number of incisors arising from one placode can be varied through local alterations of a mesenchymal signaling circuit involving β-catenin, Lef1, Tcf1 and Bmp4.     

Suppression of insulin-like3 receptor reveals the role of β-catenin and Notch signaling in gubernaculum development

During male development, the testes move from a high intraabdominal position and descend into the scrotum. The gubernaculum, an inguinoscrotal ligament connecting the testis to the lower abdomen, is believed to play a critical role in this process. The first stage of testicular descent is controlled by insulin like3 hormone (INSL3), produced in testicular Leydig cells. Deletion of Insl3 or its receptor, Rxfp2, in mice causes cryptorchidism. We produced Cre/loxP regulated shRNA transgenic mice targeting RXFP2 expression. We have shown that the transgene was able to reduce Rxfp2 gene expression and thus behaved as a hypomorphic allele of Rxfp2. Variable degrees of uni- and bilateral cryptorchidism was detected in males with the activated shRNA transgene on an Rxfp2+/- background. Conditional suppression of Rxfp2 in the gubernaculum led to cryptorchidism. Gene expression analysis of a mutant cremasteric sac using Illumina microarrays indicated abnormal expression of a significant number of genes in Wnt/β-catenin and Notch pathways. We have demonstrated profound changes in the expression pattern of β-catenin, Notch1, desmin, and androgen receptor (AR), in Rxfp2-/- male embryos, indicating the role of INSL3 in proliferation, differentiation, and survival of specific cellular components of the gubernaculum. We have shown that INSL3/RXFP2 signaling is essential for myogenic differentiation and maintenance of AR-positive cells in the gubernaculum. Males with the deletion of β-catenin or Notch1 in the gubernacular ligament demonstrated abnormal development. Our data indicates that β-catenin and Notch pathways are potential targets of INSL3 signaling during gubernacular development.     

A hydrogen bond regulates slow motions in ubiquitin by modulating a β-turn flip

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions—the interconversion of a type I into a type II β-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.     

SNX25 regulates TGF-β signaling by enhancing the receptor degradation

SNXs (sorting nexin), a family of proteins playing roles in cargo sorting and signaling from compartments within the endocytic network, regulate traffic of membrane proteins including TGF-β receptors. Here we report that the full length human and mouse SNX25, a SNX member with PX, PXA and RGS domains, co-localizes with TGF-β receptors, and forms internalized cytosolic punctae upon treatment with TGF-β. While overexpression of SNX25 inhibits TGF-β induced luciferase reporter activity, knocking down endogenous SNX25 by siRNA in NIH3T3 cells elevates the TGF-β receptor levels and facilitates TGF-β signaling. Immunoprecipitation experiments demonstrate that SNX25 interacts with TβRI. Western blot analyses indicate that SNX25 enhances the degradation of TGF-β receptors. SNX25 induced TGF-β receptor degradation is shown via the clathrin dependent endocytosis pathway into lysosome. We have characterized that PXA domain of SNX25 is required for the degradation of TβRI. Our findings demonstrate that SNX25 negatively regulates TGF-β signaling by enhancing the receptor degradation through lysosome pathway.     

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling

As a Ca²⁺ binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-β1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-β1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-β1. On treatment with TGF-β1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1. These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway. Finally, TGF-β1-induced-EMT enhanced store-operated Ca²⁺ influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-β1 on thapsigargin (TG) −induced Ca²⁺ release, but had failed to reduce store-operated Ca²⁺ influx. The alteration of intracellular Ca²⁺ concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-β1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling. Furthermore, TGF-β1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.     

Selective β2-adrenoreceptor signaling regulates osteoclastogenesis via modulating RANKL production and neuropeptides expression in osteocytic MLO-Y4 cells

The β2-adrenergic receptor (β2-AR) signaling on bone cells is the major contributor in the effect of the sympathetic nervous system on bone turnover. However, it remains unclear whether receptor activator of nuclear factor κ-Β ligand (RANKL) modulation and neuropeptides expression in osteocytes are responsible for the mechanism. This study used β2-AR stimulation to investigate cell cycle and proliferation, the gene and protein expression of RANKL, and osteoprotegerin (OPG), as well as neuropeptides regulation in osteocytic MLO-Y4 cells. Clenbuterol (CLE; a β2-AR agonist) slightly promoted the growth of MLO-Y4 cells in a concentration-dependent effect but had no effect on the proliferation index. And the concentration of 10⁻⁸ M showed a significant increase in the S-phase fraction on day 3 in comparison with the control. Additionally, CLE-promoted osteoclast formation and bone resorption in osteocytic MLO-Y4 cell-RAW264.7 cell cocultures. RANKL expression level and the ratio of RANKL to OPG in MLO-Y4 cells were enhanced in CLE treatment but were rescued by blocking β2-AR signaling. However, neuropeptide Y and α-calcitonin gene-related peptide, two neurogenic markers, were inhibited in CLE treatment of MLO-Y4 cells, which was reversed by a β2-AR blocker. The results indicate that osteocytic β2-AR plays an important role in the regulation of RANKL/OPG and neuropeptides expression, and β2-AR signaling in osteocytes can be used as a new valuable target for osteoclast-related pathologic disease.     

Mink1 regulates β-catenin-independent Wnt signaling via Prickle phosphorylation

β-Catenin-independent Wnt signaling pathways have been implicated in the regulation of planar cell polarity (PCP) and convergent extension (CE) cell movements. Prickle, one of the core proteins of these pathways, is known to asymmetrically localize proximally at the adherens junction of Drosophila melanogaster wing cells and to locally accumulate within plasma membrane subdomains in cells undergoing CE movements during vertebrate development. Using mass spectrometry, we have identified the Ste20 kinase Mink1 as a Prickle-associated protein and found that they genetically interact during the establishment of PCP in the Drosophila eye and CE in Xenopus laevis embryos. We show that Mink1 phosphorylates Prickle on a conserved threonine residue and regulates its Rab5-dependent endosomal trafficking, a process required for the localized plasma membrane accumulation and function of Prickle. Mink1 also was found to be important for the clustering of Vangl within plasma membrane puncta. Our results provide a link between Mink and the Vangl-Prickle complex and highlight the importance of Prickle phosphorylation and endosomal trafficking for its function during Wnt-PCP signaling.     

Lumican regulates osteosarcoma cell adhesion by modulating TGFβ2 activity

Human osteosarcoma cell lines were recently shown to express and secrete the small leucine rich proteoglycan (SLRP) lumican, with the ability to regulate the growth and motility of these cells. In this study, lumican-deficient Saos 2 cells were demonstrated to have increased adhesive capability onto fibronectin (FN) (p≤0.01). Upon neutralization of endogenous transforming growth factor β2 (TGF-β2) activity, no difference in the ability of lumican siRNA-transfected and scramble siRNA-transfected Saos 2 cells to adhere onto FN was detected (p=NS). Exogenous TGF-β2 was shown to stimulate Saos 2 cell adhesion to FN (p≤0.01). These results therefore, suggest that the inverse correlation existing between lumican expression and Saos 2 cell adhesion is dependent on active TGF-β2 signaling. Furthermore, the significant increase in Smad 2 activation present in lumican-deficient cells (p≤0.01) was annulled in the presence of the anti-TGF-β2 peptide, demonstrating that lumican is an upstream regulator of the TGF-β2/Smad 2 signaling cascade. Crucial to FN-dependent adhesion, β1 integrin expression and pFAK activation were likewise identified as downstream TGF-β2 effectors regulated by lumican expression. In conclusion, this study demonstrates a novel out-in signaling circuit in human osteosarcoma cells: secreted to extracellular matrix lumican is an endogenous inhibitor of TGF-β2 activity, resulting in downstream effector modulation including pSmad 2, integrin β1 and pFAK to regulate osteosarcoma adhesion.