Michel L. Tremblay

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B.

Common obesity is primarily characterized by resistance to the actions of the hormone leptin. Mice deficient in protein tyrosine phosphatase 1B (PTP1B) are resistant to diabetes and diet-induced obesity, prompting us to further define the relationship between PTP1B and leptin in modulating obesity. Leptin-deficient (Lep(ob/ob)) mice lacking PTP1B exhibit an attenuated weight gain, a decrease in adipose tissue, and an increase in resting metabolic rate. Furthermore, PTP1B-deficient mice show an enhanced response toward leptin-mediated weight loss and suppression of feeding. Hypothalami from these mice also display markedly increased leptin-induced Stat3 phosphorylation. Finally, substrate-trapping experiments demonstrate that leptin-activated Jak2, but not Stat3 or the leptin receptor, is a substrate of PTP1B. These results suggest that PTP1B negatively regulates leptin signaling, and provide one mechanism by which it may regulate obesity.

Inside the human cancer tyrosine phosphatome

Members of the protein tyrosine phosphatase (Ptp) family dephosphorylate target proteins and counter the activities of protein tyrosine kinases that are involved in cellular phosphorylation and signalling. As such, certain PTPs might be tumour suppressors. Indeed, PTPs play an important part in the inhibition or control of growth, but accumulating evidence indicates that some PTPs may exert oncogenic functions. Recent large-scale genetic analyses of various human tumours have highlighted the relevance of PTPs either as putative tumour suppressors or as candidate oncoproteins. Progress in understanding the regulation and function of PTPs has provided insights into which PTPs might be potential therapeutic targets in human cancer.

The T Cell Protein Tyrosine Phosphatase Is a Negative Regulator of Janus Family Kinases 1 and 3

BACKGROUND The immune response is regulated through a tightly controlled cytokine network. The counteracting balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activity regulates intracellular signaling in the immune system initiated by these extracellular polypeptides. Mice deficient for the T cell protein tyrosine phosphatase (TCPTP) display gross defects in the hematopoietic compartment, indicating a critical role for TCPTP in the regulation of immune homeostasis. To date, the molecular basis underlying this phenotype has not been reported. RESULTS We have identified two members of the Janus family of tyrosine kinases (JAKs), JAK1 and JAK3, as bona fide substrates of TCPTP. Inherent substrate specificity in the TCPTP-JAK interaction is demonstrated by the inability of other closely related PTP family members to form an in vivo interaction with the JAKs in hematopoietic cells. In keeping with a negative regulatory role for TCPTP in cytokine signaling, expression of TCPTP in T cells abrogated phosphorylation of STAT5 following interleukin (IL)-2 stimulation. TCPTP-deficient lymphocytes treated with IL-2 had increased levels of tyrosine-phosphorylated STAT5, and thymocytes treated with interferon (IFN)-alpha or IFN-gamma had increased tyrosine-phosphorylated STAT1. Hyperphosphorylation of JAK1 and elevated expression of iNOS was observed in IFN-gamma-treated, TCPTP-deficient, bone marrow-derived macrophages. CONCLUSIONS We have identified JAK1 and JAK3 as physiological substrates of TCPTP. These results indicate a negative regulatory role for TCPTP in cytokine signaling and provide insight into the molecular defect underlying the phenotype of TCPTP-deficient animals.

Antitumor Activity and Mechanism of Action of the Cyclopenta[b]benzofuran, Silvestrol

Background Flavaglines are a family of natural products from the genus Aglaia that exhibit anti-cancer activity in vitro and in vivo and inhibit translation initiation. They have been shown to modulate the activity of eIF4A, the DEAD-box RNA helicase subunit of the eukaryotic initiation factor (eIF) 4F complex, a complex that stimulates ribosome recruitment during translation initiation. One flavagline, silvestrol, is capable of modulating chemosensitivity in a mechanism-based mouse model. Methodology/Principal Findings Among a number of flavagline family members tested herein, we find that silvestrol is the more potent translation inhibitor among these. We find that silvestrol impairs the ribosome recruitment step of translation initiation by affecting the composition of the eukaryotic initiation factor (eIF) 4F complex. We show that silvestrol exhibits significant anticancer activity in human breast and prostate cancer xenograft models, and that this is associated with increased apoptosis, decreased proliferation, and inhibition of angiogenesis. We demonstrate that targeting translation by silvestrol results in preferential inhibition of weakly initiating mRNAs. Conclusions/Significance Our results indicate that silvestrol is a potent anti-cancer compound in vivo that exerts its activity by affecting survival pathways as well as angiogenesis. We propose that silvestrol mediates its effects by preferentially inhibiting translation of malignancy-related mRNAs. Silvestrol appears to be well tolerated in animals.

Protein-tyrosine Phosphatase 1B Potentiates IRE1 Signaling during Endoplasmic Reticulum Stress

Protein-tyrosine phosphatase 1B (PTP-1B) is the prototypic tyrosine phosphatase whose function in insulin signaling and metabolism is well established. Although the role of PTP-1B in dephosphorylating various cell surface receptor tyrosine kinases is clear, the mechanisms by which it modulates receptor function from the endoplasmic reticulum (ER) remains an enigma. Here, we provide evidence that PTP-1B has an essential function in regulating the unfolded protein response in the ER compartment. The absence of PTP-1B caused impaired ER stress-induced IRE1 signaling. More specifically, JNK activation, XBP-1 splicing, and EDEM (ER degradation-enhancing α-mannosidase-like protein) gene induction, as well as ER stress-induced apoptosis, were attenuated in PTP-1B knock-out mouse embryonic fibroblasts in response to two ER stressors, tunicamycin and azetidine-2 carboxylic acid. We demonstrate that PTP-1B is not just a passive resident of the ER but on the contrary has an essential role in potentiating IRE1-mediated ER stress signaling pathways.

Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase σ

Protein tyrosine phosphatase σ (PTP-σ, encoded by the Ptprs gene) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases that is highly expressed during mammalian embryonic development in the germinal cell layer lining the lateral ventricles of the developing brain, dorsal root ganglia, Rathkes pouch, olfactory epithelium, retina and developing lung and heart. On the basis of its expression and homology with the Drosophila melanogaster orthologues DPTP99 and DPTP100A (Refs 5,6), which have roles in the targeting of axonal growth cones, we hypothesized that PTP-σ may also have a modulating function in cell-cell interactions, as well as in axon guidance during mammalian embryogenesis. To investigate its function in vivo, we generated Ptprs-deficient mice. The resulting Ptprs-/- animals display retarded growth, increased neonatal mortality, hyposmia and hypofecundity. Anatomical and histological analyses showed a decrease in overall brain size with a severe depletion of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cells in Ptprs-/- hypothalamus. Ptprs-/- mice have an enlarged intermediate pituitary lobe, but smaller anterior and posterior lobes. These results suggest that tyrosine phosphorylation-dependent signalling pathways regulated by PTP-σ influence the proliferation and/or adhesiveness of various cell types in the developing hypothalamo-pituitary axis.

Leishmania GP63 Alters Host Signaling Through Cleavage-Activated Protein Tyrosine Phosphatases

The parasite protein GP63 triggers cleavage and activation of host protein tyrosine phosphatases to promote infection. Exploiting the Host’s Phosphatases Leishmaniasis is a globally important infectious disease caused by the parasite Leishmania. Gomez et al. show that infection of macrophages with Leishmania alters the activity of multiple protein tyrosine phosphatases (PTPs) through cleavage mediated by the parasite protein GP63. The activated PTPs inhibit macrophage inflammatory immune responses through dephosphorylation of Janus kinases. In addition to the PTP SHP-1, previously reported to be activated in response to Leishmania infection, Gomez et al. show that the PTPs TCPTP and PTP1B are also activated and that PTP1B serves a key role in the initial stages of disease progression in mice. With more than 12 million people affected worldwide, 2 million new cases occurring per year, and the rapid emergence of drug resistance and treatment failure, leishmaniasis is an infectious disease for which research on drug and vaccine development, host-pathogen, and vector-parasite interactions are current international priorities. Upon Leishmania-macrophage interaction, activation of the protein tyrosine phosphatase (PTP) SHP-1 rapidly leads to the down-regulation of Janus kinase and mitogen-activated protein kinase signaling, resulting in the attenuation of host innate inflammatory responses and of various microbicidal macrophage functions. We report that, in addition to SHP-1, the PTPs PTP1B and TCPTP are activated and posttranslationally modified in infected macrophages, and we identify an essential role for PTP1B in the in vivo progression of Leishmania infection. The mechanism underlying PTP modulation involves the proteolytic activity of the Leishmania surface protease GP63. Access of GP63 to macrophage PTP1B, TCPTP, and SHP-1 is mediated in part by a lipid raft–dependent mechanism, resulting in PTP cleavage and stimulation of phosphatase activity. Collectively, our data present a mechanism of cleavage-dependent activation of macrophage PTPs by an obligate intracellular pathogen and show that internalization of GP63, a key Leishmania virulence factor, into host macrophages is a strategy the parasite uses to interact and survive within its host.

Selective regulation of tumor necrosis factor-induced Erk signaling by Src family kinases and the T cell protein tyrosine phosphatase.

The proinflammatory cytokine tumor necrosis factor (TNF) modulates cellular responses through the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways, but the molecular mechanisms underlying MAPK activation are unknown. T cell protein tyrosine phosphatase (TCPTP) is essential for hematopoietic development and negatively regulates inflammatory responses. Using TCPTP-deficient fibroblasts, we show here that TCPTP regulates TNF-induced MAPK but not NF-κB signaling. TCPTP interacted with the adaptor protein TRAF2, and dephosphorylated and inactivated Src tyrosine kinases to suppress downstream signaling through extracellular signal–regulated kinases and production of interleukin 6. These results link TCPTP and Src tyrosine kinases to the selective regulation of TNF-induced MAPK signaling and identify a previously unknown mechanism for modulating inflammatory responses mediated by TNF.

PHOSPHATASES IN CELL-MATRIX ADHESION AND MIGRATION

Many proteins that have been implicated in cell–matrix adhesion and cell migration are phosphorylated, which regulates their folding, enzymatic activities and protein–protein interactions. Although modulation of cell motility by kinases is well known, increasing evidence confirms that phosphatases are essential at each stage of the migration process. Phosphatases can control the formation and maintenance of the actin cytoskeleton, regulate small GTPase molecular switches, and modulate the dynamics of matrix–adhesion interaction, actin contraction, rear release and migratory directionality.