Transglutaminase Type 2 is Involved in the Hematopoietic Stem Cells Homeostasis.

Type 2 transglutaminase (TG2) is a multifunctional protein involved in various biological processes playing a key regulatory role in cell homeostasis such as cell death and autophagy. New evidence is emerging that support an important role of autophagy in regulating normal hematopoiesis. Prompted by these findings, in this study we investigated in vivo involvement of TG2 in mouse hematopoiesis under normal or nutrient deprivation conditions. We found that the number and rate of differentiation of bone marrow hematopoietic stem cell was decreased in the TG2 knockout mice. We present evidence showing that these effects on hematopoietic system are very likely due to the TG2-dependent impairment of autophagy. In fact, stimulation of autophagy by starvation is able to rescue the block of the differentiation of stem cells progenitors in the TG2 KO mice. It was also shown that the RhoA/ERK½ pathway, known to be essential for regulation of the bone marrow progenitor cells homeostasis, was significantly impaired in the absence of TG2. Hence, this study expanded our knowledge about TG2 discovering a role of this enzyme in regulation of hematopoiesis.

DNA damage-induced phosphatase Wip1 in regulation of hematopoiesis, immune system and inflammation.

PP2C serine-threonine phosphatase, Wip1, is an important regulator of stress response. Wip1 controls a number of critical cellular functions: proliferation, cell cycle arrest, senescence and programmed cell death, apoptosis or autophagy. Ppm1d, the gene encoding Wip1 phosphatase, is expressed in hematopoietic progenitors, stem cells, neutrophils, macrophages B and T lymphocytes in bone marrow and peripheral blood. The Wip1-/- mice display immunodeficiency, abnormal lymphoid histopathology in thymus and spleen, defects in B- and T-cell differentiation, as well as susceptibility to viral infection. At the same time, Wip1 knockout mice exhibit pro-inflammatory phenotype in skin and intestine in the model of inflammatory bowel disease (IBD) with elevated levels of inflammation-promoting cytokines TNF-α, IL-6, IL-12, IL-17. Several Wip1 downstream targets can mediate Wip1 effects on hematopoietic system including, p53, ATM, p38MAPK kinase, NFkB, mTOR. Here, we summarized the current knowledge on the role of Wip1 in the differentiation of various hematopoietic lineages and how Wip1 deficiency affects the functions of immune cells.

ROLE OF Wip1-p53 AXIS IN RESPONSE OF MURINE CELLS TO TREATMENT WITH SODIUM BUTYRATE AND MEK/ERK SIGNALLING PATHWAY INHIBITOR.

The use of histone deacetylase inhibitors and inhibitors of MEK/ERK-pathway is proposed as a novel potential approach in cancer treatment. Here we studied the effects of histone deacetylase inhibitor, sodium butyrate, and MEK/ERK-pathway inhibitor, PD0325901, on cells with modifications in genes involved in anti-cancer therapy response, Wip1 phosphatase and p53. We have investigated the effect of these agents on cell cycle of wild-type cells, Wip1 knockout cells and cells with double deletion of Wip1 and p53. Our results showed that more severe changes in S and G2/M phases were observed in response to sodium butyrate in Wip1-defecient cells than in wild-type cells. Meanwhile, PD0325901 treatment led to G1 arrest. At the same time, a «sodium butyrate type¼ response dominated the response to combined treatment with both drugs in Wip1-deficient cells, while the response of Wip1­/­/p53­/­ cells to combined treatment was similar to the single use of PD0325901. Wip1­/­ and Wip1­/­/p53­/­ cells were more sensitive to the use of PD0325901 than wild-type cells. Obtained results suggest that Wip1 deficiency sensitizes cells to sodium butyrate and to MEK/ERK inhibitors independently from Wip1 main target protein ­ p53. Data acquired give insights into role of Wip1 in cellular responses to treatment with HDAC and MEK/ERK inhibitors.

HSP110 promotes colorectal cancer growth through STAT3 activation.

Heat shock protein 110 (HSP110) is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps cells survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently shown that colorectal cancer patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110-inactivating mutation (HSP110DE9). In this work, we used patient biopsies, human colorectal cancer cells grown in vitro and in vivo (xenografts), and intestinal crypts to demonstrate that HSP110 is also involved in colon cancer growth. We showed that HSP110 induces colon cancer cell proliferation and that this effect is associated with STAT3 activation, specifically an increase in STAT3 phosphorylation, nuclear translocation and transcription factor activity. STAT3 inhibition blocks the proliferative effect of HSP110. From a molecular standpoint, we demonstrated that HSP110 directly binds to STAT3, thereby facilitating its phosphorylation by JAK2. Finally, we showed a correlation between HSP110 expression and STAT3 phosphorylation in colon cancer patient samples. Thus, the expression of HSP110 in colon cancer contributes to STAT3-dependent tumor growth and the frequent inactivating mutation of this chaperone is probably an important event underlying the improved prognosis in colon cancer displaying MSI.

Wee1 inhibition potentiates Wip1-dependent p53-negative tumor cell death during chemotherapy.

Inactivation of p53 found in more than half of human cancers is often associated with increased tumor resistance to anti-cancer therapy. We have previously shown that overexpression of the phosphatase Wip1 in p53-negative tumors sensitizes them to chemotherapeutic agents, while protecting normal tissues from the side effects of anti-cancer treatment. In this study, we decided to search for kinases that prevent Wip1-mediated sensitization of cancer cells, thereby interfering with efficacy of genotoxic anti-cancer drugs. To this end, we performed a flow cytometry-based screening in order to identify kinases that regulated the levels of γH2AX, which were used as readout. Another criterion of the screen was increased sensitivity of p53-negative tumor cells to cisplatin (CDDP) in a Wip1-dependent manner. We have found that a treatment with a low dose (75 nM) of MK-1775, a recently described specific chemical inhibitor of Wee1, decreases CDDP-induced H2AX phosphorylation in p53-negative cells and enhances the Wip1-sensitization of p53-negative tumors. We were able to reduce CDDP effective concentration by 40% with a combination of Wip1 overexpression and Wee1 kinase inhibition. We have observed that Wee1 inhibition potentiates Wip1-dependent tumor sensitization effect by reducing levels of Hipk2 kinase, a negative regulator of Wip1 pathway. In addition, during CDDP treatment, the combination of Wee1 inhibition and Wip1 overexpression has a mild but significant protective effect in normal cells and tissues. Our results indicate that inhibition of the negative regulators of Wip1 pathway, Wee1 and Hipk2, in p53-negative tumors could potentiate efficiency of chemotherapeutic agents without concomitant increase of cytotoxicity in normal tissues. The development and clinical use of Wee1 and Hipk1 kinase chemical inhibitors might be a promising strategy to improve anti-cancer therapy.

The HSP90 inhibitor, 17AAG, protects the intestinal stem cell niche and inhibits graft versus host disease development.

Graft versus host disease (GvHD), which is the primary complication of allogeneic bone marrow transplantation, can alter the intestinal barrier targeted by activated donor T-cells. Chemical inhibition of the stress protein HSP90 was demonstrated in vitro to inhibit T-cell activation and to modulate endoplasmic reticulum (ER) stress to which intestinal cells are highly susceptible. Since the HSP90 inhibitor 17-allylamino-demethoxygeldanamycin (17AAG) is developed in clinics, we explored here its ability to control intestinal acute GvHD in vivo in two mouse GvHD models (C57BL/6BALB/c and FVB/NLgr5-eGFP), ex vivo in intestine organoids and in vitro in intestinal epithelial cultures. We show that 17AAG decreases GvHD-associated mortality without impairing graft versus leukemia effect. While 17AAG effect in T-cell activation is just moderate at the dose used in vivo, we observe a striking intestinal integrity protection. At the intestine level, the drug promotes the splicing of the transcription factor X-box binding protein 1 (XBP1), which is a key component of the ER stress. This effect is associated with a decrease in intestinal damage and an increase in Lgr5(+) stem cells, Paneth cells and defensins production. The importance of XBP1 splicing control is further confirmed in cultured cells and organoids of primary intestinal epithelium where XBP1 is either shRNA depleted or inhibited with toyocamycin. In conclusion, 17AAG has a protective effect on the epithelial intestinal barrier in mouse models of acute GvHD. This compound deserves to be tested in the therapeutic control of acute GvHD.

Role of Gadd45a in Wip1-dependent regulation of intestinal tumorigenesis.

Conversion of intestinal stem cells into tumor-initiating cells is an early step in Apc(Min)-induced polyposis. Wild-type p53-induced phosphatase 1 (Wip1)-dependent activation of a DNA damage response and p53 has a permanent role in suppression of stem cell conversion, and deletion of Wip1 lowers the tumor burden in Apc(Min) mice. Here we show that cyclin-dependent kinase inhibitor 2a, checkpoint kinase 2, and growth arrest and DNA damage gene 45a (Gadd45a) exert critical functions in the tumor-resistant phenotype of Wip1-deficient mice. We further identified Gadd45a as a haploinsufficient gene in the regulation of Wip1-dependent tumor resistance in mice. Gadd45a appears to function through its ability to activate the Jnk-dependent signaling pathway that in turn is a necessary mediator of the proapoptotic functions of p53 that respond to activation of the ß-catenin signaling pathway. We propose that silencing of Gadd45a is sufficient to override p53 activation in the presence of active ß-catenin under conditions of an enhanced DNA damage response.

The role of the MKK6/p38 MAPK pathway in Wip1-dependent regulation of ErbB2-driven mammary gland tumorigenesis.

There is increasing evidence for the role of wild-type p53 induced phosphatase 1 (Wip1) phosphatase in the regulation of tumorigenesis. To evaluate Wip1 as a breast cancer oncogene, we generated a mouse strain with targeted expression of Wip1 to the breast epithelium. We found that these mice are prone to cancer when intercrossed with transgenics expressing the ErbB2 oncogene but not conditional knockouts for Brca2. This tumor-prone phenotype of Wip1 is fully eliminated through attenuation of proliferation by activating the MKK6/p38 mitogen-activated protein kinases (MAPK) cascade in mice bearing a constitutively active form of MKK6. We propose that Wip1 phosphatase operates within the MKK6/p38 MAPK signaling pathway to promote ErbB2-driven mammary gland tumorigenesis.

Heat shock proteins in cardiosurgery patients.

OBJECTIVE: Cytoplasmic members of the heat shock protein HSP70, family, inducible HSP72 and constitutive HSC73, are known to protect cells and organisms against harmful factors including ischemia, trauma, etc. The up-regulation of HSP70 was shown to greatly increase resistance of myocardial cells in vitro as well as in transgenic animals. It seems reasonable to expect that in patients undergoing open heart surgery cytoplasmic HSP70 should play a protective role, reducing the risk of the myocardial cell injury. METHODS: Using Western blotting, we determined levels of HSP72 and HSC73 in myocardium and peripheral blood lymphocytes of 51 patients with coronary and valvular diseases. In all the cases, HSP70 was detected in samples of the right atria before and after cardiopulmonary bypass. RESULTS: Induction of HSP72 was observed in 40% of all patients and correlated with the endurance of cardiopulmonary bypass and with disease duration in 33 patients with coronary artery disease. The cardioprotective effect of the elevated pre-operational level of HSP72 was shown to correlate with the lower activity of cardiospecific enzymes in the coronary disease patients. The HSC73 level in the right atria did not depend on conditions of the open heart surgery, while in some cases, it was increased after bypass. No correlation has been found between preoperational content of HSP72/HSC73 in lymphocytes and its pre- or post-bypass content in myocardium. CONCLUSION: HSP72 is implicated in cardioprotection in combination with some other factors, and its pre-operational level, among other parameters, might be of prognostic value.

[Role of heat-shock proteins in cardioprotection of patients operated on for ischemic heart disease]. / Rol' belka teplovogo shoka v kardioprotektsii u bol'nykh, operirovannykh po povodu ishemicheskoi bolezni serdtsa.

Major stress protein HSP72 is known to participate in protecting cells and organisms against harmful factors including ischemia, trauma etc. Under study was the level of HSP72 in the myocardium of 32 patients with coronary disease operated in Military-medical academy. HSP72 was detected in probes of the right atria before and after pre-cardiopulmonary bypass in all cases induction of HSP72 was observed in 40% of patients, and directly correlated with the time of cardiopulmonary bypass and standing of the disease. The cardioprotective effect of the elevated pre-operational level of HSP72 was shown to be proportionate to the lower activity of cardiospecific enzymes, creatine phosphokinase (CK-MB). It is suggested that HSP72 is involved in the mechanism of cardioprotection during cardiopulmonary bypass.