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Plasmacytoid dendritic cells (pDCs) promote viral elimination by producing large amounts of Type I interferon. Recent studies have shown that pDCs regulate the pathogenesis of diverse inflammatory diseases, such as cancer. Fatty acid-binding protein 5 (FABP5) is a cellular chaperone of long-chain fatty acids that induce biological responses. Although the effects of FABP-mediated lipid metabolism are well studied in various immune cells, its role in pDCs remains unclear. This study, which compares wild-type and Fabp5 -/- mice, provides the first evidence that FABP5-mediated lipid metabolism regulates the commitment of pDCs to inflammatory vs tolerogenic gene expression patterns in the tumor microenvironment and in response to toll-like receptor stimulation. Additionally, we demonstrated that FABP5 deficiency in pDCs affects the surrounding cellular environment, and that FABP5 expression in pDCs supports the appropriate generation of regulatory T cells (Tregs). Collectively, our findings reveal that pDC FABP5 acts as an important regulator of tumor immunity by controlling lipid metabolism., (© 2021 UICC.)

Fatty acid-binding protein (FABP) 5 is highly expressed in various types of tumors and is strongly correlated with tumor growth, development, and metastasis. However, it is unclear how the expression of FABP5 in the host affects tumor progression. In this study, using a lung tumor metastasis model in mice, we found that FABP5-deficient mice were more susceptible to tumor metastasis, which is accompanied by infiltration of a lower frequency of activated natural killer (NK) cells in the lung. Additionally, FABP5 deficiency leads to impaired maturation of NK cells in the lungs, but not in the bone marrow and spleen. Taken together, our results provide the first evidence that FABP5 in the host regulates lung tumor metastasis through controlling NK cell maturation., (© 2021 Federation of European Biochemical Societies.)

Dietary obesity is regarded as a problem worldwide, and it has been revealed the strong linkage between obesity and allergic inflammation. Fatty acid-binding protein 5 (FABP5) is expressed in lung cells, such as alveolar epithelial cells (ECs) and alveolar macrophages, and plays an important role in infectious lung inflammation. However, we do not know precise mechanisms on how lipid metabolic change in the lung affects allergic lung inflammation. In this study, we showed that Fabp5 -/- mice exhibited a severe symptom of allergic lung inflammation. We sought to examine the role of FABP5 in the allergic lung inflammation and demonstrated that the expression of FABP5 acts as a novel positive regulator of ST2 expression in alveolar ECs to generate retinoic acid (RA) and supports the synthesis of RA from type II alveolar ECs to suppress excessive activation of innate lymphoid cell (ILC) 2 during allergic lung inflammation. Furthermore, high-fat diet (HFD)-fed mice exhibit the downregulation of FABP5 and ST2 expression in the lung tissue compared with normal diet (ND)-fed mice. These phenomena might be the reason why obese people are more susceptible to allergic lung inflammation. Thus, FABP5 is potentially a therapeutic target for treating ILC2-mediated allergic lung inflammation.

Lipids are major molecules for the function of organisms and are involved in the pathophysiology of various diseases. Fatty acids (FAs) signaling and their metabolism are some of the most important pathways in tumor development, as lipids serve as energetic sources during carcinogenesis. Fatty acid binding proteins (FABPs) facilitate FAs transport to different cell organelles, modulating their metabolism along with mediating other physiological activities. FABP7, brain-typed FABP, is thought to be an important molecule for cell proliferation in healthy as well as diseased organisms. Several studies on human tumors and tumor-derived cell lines put FABP7 in the center of tumorigenesis, and its high expression level has been reported to correlate with poor prognosis in different tumor types. Several types of FABP7-expressing tumors have shown an up-regulation of cell signaling activity, but molecular mechanisms of FABP7 involvement in tumorigenesis still remain elusive. In this review, we focus on the expression and function of FABP7 in different tumors, and possible mechanisms of FABP7 in tumor proliferation and migration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fatty-acid-binding proteins (FABPs) are key intracellular molecules involved in the uptake, transportation and storage of fatty acids and in the mediation of signal transduction and gene transcription. However, little is known regarding their expression and function in the oligodendrocyte lineage. We evaluate the in vivo and in vitro expression of FABP5 and FABP7 in oligodendrocyte lineage cells in the cortex and corpus callosum of adult mice, mixed cortical culture and oligosphere culture by immunofluorescent counter-staining with major oligodendrocyte lineage markers. In all settings, FABP7 expression was detected in NG2(+)/PDGFRα(+) oligodendrocyte progenitor cells (OPCs) that did not express FABP5. FABP5 was detected in mature CC1(+)/MBP(+) oligodendrocytes that did not express FABP7. Analysis of cultured OPCs showed a significant decrease in the population of FABP7-knockout (KO) OPCs and their BrdU uptake compared with wild-type (WT) OPCs. Upon incubation of OPCs in oligodendrocyte differentiation medium, a significantly lower percentage of FABP7-KO OPCs differentiated into O4(+) oligodendrocytes. The percentage of mature MBP(+) oligodendrocytes relative to whole O4(+)/MBP(+) oligodendrocytes was significantly lower in FABP7-KO and FABP5-KO than in WT cell populations. The percentage of terminally mature oligodendrocytes with membrane sheet morphology was significantly lower in FABP5-KO compared with WT cell populations. Thus, FABP7 and FABP5 are differentially expressed in oligodendrocyte lineage cells and regulate their proliferation and/or differentiation. Our findings suggest the involvement of FABP7 and FABP5 in the pathophysiology of demyelinating disorders, neuropsychiatric disorder and glioma, conditions in which OPCs/oligodendrocytes play central roles.

Bortezomib is an effective agent for treating multiple myeloma (MM). To investigate the underlying mechanisms associated with acquired resistance to this agent, we established two bortezomib-resistant MM cell lines, KMS-11/BTZ and OPM-2/BTZ, the 50% inhibitory concentration values of which were respectively 24.7- and 16.6-fold higher than their parental cell lines. No activation of caspase and BH3-only proteins such as Noxa was noted in bortezomib-resistant cells after exposure to the drug. The accumulation of polyubiquitinated proteins was reduced in bortezomib-resistant cells compared with the parental cells, associated with avoidance of catastrophic ER stress as assessed by downregulation of CHOP expression. These resistant MM cells have a unique point mutation, G322A, in the gene encoding the proteasome beta5 subunit (PSMB5), likely resulting in conformational changes to the bortezomib-binding pocket of this subunit. KMS-11 parental cells transfected to express mutated PSMB5 also showed reduced bortezomib-induced apoptosis compared with those expressing wild-type PSMB5 or the parental cells. Expression of mutated PSMB5 was associated with the prevention of the accumulation of unfolded proteins. Thus, a fraction of MM cells may acquire bortezomib resistance by suppressing apoptotic signals through the inhibition of unfolded protein accumulation and subsequent excessive ER stress by a mutation of the PSMB5 gene.

Previous work from our laboratory has demonstrated overexpression of chemokines in head and neck cancer and the utility of targeting these proteins for tumor therapy in a preclinical model. However, the mechanisms involved are unexplored. Through gene expression analysis, we found that expression of vascular endothelial growth factor (VEGF-C) was elevated in HN12 cells expressing high levels of CXCL5. In the present study, we have investigated the contribution of VEGF-C to tumor cell growth and motility. RNAi-mediated knockdown of VEGF-C expression in HN12 cells, which express high levels of CXCL5, resulted in a decrease in proliferation. Conversely, forced expression of VEGF-C in HN4 tumor cells with low endogenous CXCL5 levels increased cell growth. Suppression of VEGF-C inhibited migration of HN12 cells. Similarly, HN4 cells showed reduced migration towards conditioned media collected from HN12 cells with VEGF-C knockdown compared to controls, while HN4/VEGF-C conditioned media stimulated cell migration. Moreover, tumor growth in vivo was markedly reduced when VEGF-C expression was blocked by shRNA. Finally, determination of VEGF-C expression in squamous carcinoma cell lines revealed universal overexpression compared to normal keratinocytes. These findings support a role for VEGF-C in head and neck squamous cell carcinogenesis., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Previous work from our laboratory has demonstrated overexpression of chemokines in head and neck cancer, and the utility of targeting CXCL5 for tumor therapy in a preclinical model. In the present study, we investigated the contribution of a related chemokine, CXCL8, to cellular properties associated with tumor progression, namely cell growth and motility. Expression of CXCL8 was detectable in multiple squamous carcinoma cell lines, indicating a possible role in pathogenesis. Overexpression of CXCL8 in HN4 primary tumor cells with low endogenous CXCL8 levels was found to increase cell growth, as judged by cell counting and MTT assays. Conversely, RNAi-mediated knockdown of CXCL8 expression in HN12 cells, derived from a synchronous metastasis and which express high levels of this chemokine, resulted in a decrease in proliferation. Similarly, overexpression of CXCL8 enhanced migration of HN4 cells, while suppression of CXCL8 inhibited HN12 cell migration and invasion through a basement membrane substitute. Taken together, these findings support the hypothesis that CXCL8 affects multiple processes involved in tumor progression and identify CXCL8 as a potential therapeutic target, similar to CXCL5.

The biological properties of squamous carcinoma cells are intimately regulated by a multitude of cytokines and growth factors; the most well studied of these include epidermal growth factor receptor agonists and members of the transforming growth factor-beta family. The recent explosion of research in the field of chemokine function as a mediator of tumor progression has led to the possibility that these small, immunomodulatory proteins also play key roles in squamous carcinogenesis and may, therefore, be potential targets for novel therapeutic approaches.

One Japanese pedigree of familial essential thrombocythemia (FET) inherited in an autosomal-dominant manner is presented. A unique point mutation, serine 505 to asparagine 505 (Ser505Asn), was identified in the transmembrane domain of the c-MPL gene in all of the 8 members with thrombocythemia, but in none of the other 8 unaffected members in this FET family. The Ba/F3 cells expressing the mutant Asn505 acquired interleukin 3 (IL-3)-independent survival capacity, whereas those expressing wild-type Ser505 did not. The autonomous phosphorylation of Mek1/2 and Stat5b was observed in the mutant Ba/F3 cells in the absence of IL-3. The former was also found in platelets derived from the affected individual in the absence of thrombopoietin. These results show that the Asn505 is an activating mutation with respect to the intracellular signaling and survival of the cells. This is the first report of FET deriving from a dominant-positive activating mutation of the c-MPL gene.

Objective: To examine the prevalence, clinical associations, and pathogenic role of autoantibodies to c-Mpl, the thrombopoietin (TPO) receptor, in patients with systemic lupus erythematosus (SLE)., Methods: Sera from 69 SLE patients, 84 patients with idiopathic thrombocytopenic purpura (ITP), and 60 healthy individuals were screened for anti-c-Mpl antibodies by enzyme-linked immunosorbent assay using recombinant c-Mpl as an antigen. Clinical findings, autoantibody profiles, and serum TPO levels were compared between SLE patients with and without anti-c-Mpl antibodies. A pathogenic role for the anti-c-Mpl antibody was evaluated by examining its inhibitory effect on TPO-dependent cell proliferation and megakaryocyte colony formation., Results: Serum anti-c-Mpl antibody was detected in 8 SLE patients (11.6%) and 7 ITP patients (8.3%), but in none of the healthy controls. Anti-c-Mpl antibody was associated with thrombocytopenia (P = 0.0002) and a decrease in bone marrow megakaryocytes (P = 0.02) in SLE patients. Serum TPO levels in thrombocytopenic SLE patients with anti-c-Mpl antibodies were significantly elevated compared with levels in those without the antibodies (P = 0.007). IgG fractions purified from anti-c-Mpl antibody-positive sera bound to c-Mpl expressed on the cell surface and inhibited TPO-dependent cell proliferation and megakaryocyte colony formation., Conclusion: Autoantibody to c-Mpl is present in a subset of SLE patients with thrombocytopenia and megakaryocytic hypoplasia. It is likely that the impaired thrombopoiesis in these patients is mediated by the anti-c-Mpl antibody, which functionally blocks an interaction between TPO and c-Mpl.

We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU-Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood-derived CD34(+)IL-6R(-) cells as a target. TPO alone induced a significant number of CFU-Meg colonies. Although stromal cell-derived factor-1 (SDF-1) or SCF alone did not support CFU-Meg colony formation, these factors had a synergistic effect on CFU-Meg colony formation in the presence of TPO. The combination of SDF-1, SCF and TPO induced twice as many CFU-Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU-Meg colony formation. LY294002 and GF109203X (inhibitors of PI3-K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU-Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF-1, and TPO + SCF + SDF-1, whereas it abolished the effect of SDF-1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c-mpl, c-kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU-Meg, and SDF-1 is a potentiator of human megakaryocytopoiesis.

Thrombopoietin (TPO), or megakaryocyte growth and development factor (MGDF), has been shown to potentiate the sensitivity of normal human platelets to various agonists in vitro. The present study investigated the functional and biochemical properties of platelets from mice rendered thrombocytopenic by sublethal irradiation with regard to the reactivity to recombinant murine MGDF (rmMGDF) in vitro. During the course of reversible thrombocytopenia following irradiation, platelets from irradiated mice which had lower platelet counts and reciprocally higher plasma TPO levels showed lower reactivity to rmMGDF in agonist-induced platelet aggregation. Intravenous injections of recombinant soluble murine c-Mpl (sMpl), which has the ability to capture TPO, after irradiation restored the reactivity of platelets at the platelet nadir to rmMGDF. On the other hand, platelets prepared from normal mice 3 h after a single intravenous injection of pegylated rmMGDF did not respond to rmMGDF. There was a marked decrease in c-Mpl and Janus kinase 2 (JAK2) in platelets from irradiated mice at the platelet nadir. Similar results were observed with platelets from mice administered pegylated rmMGDF. JAK2 was only moderately decreased, however, in platelets from mice given sMpl after irradiation. These results indicate that exposure of platelets to increased endogenous TPO levels in vivo in thrombocytopenic mice leads to a reduction in the platelet reactivity to rmMGDF in vitro. Further, these results suggest that the c-Mpl-mediated signaling pathway, which is essential for the priming effect of rmMGDF, is defective in thrombocytopenic murine platelets.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.

The regulation mechanism of circulating thrombopoietin (TPO) level in human newborns remains unknown. In the present study, we examined whether the TPO concentrations in cord blood were influenced by the difference in the delivery method and the presence or absence of maternal/fetal complications. Cortisol levels were simultaneously measured to assess the adrenal response of fetuses. Both the TPO level and the cortisol level were substantially greater in the neonates delivered vaginally with and without the complications than in those delivered by cesarean section without the complications. The binding assay showed that the incubation of mpl(+)/BaF3 cells with cortisol gave rise to a significant decrease in the binding sites of TPO. These results suggest that the stress to the fetuses near the time of delivery affects the cord blood TPO levels, which may be mediated in part by the action of cortisol on the TPO-mpl binding system.

Transient myeloproliferative disorder (TMD) in neonates with Down syndrome is characterized by increased megakaryoblastic cells in the peripheral blood. Despite their spontaneous regression in weeks, prognosis is not always favorable because of fatal hepatic fibrosis. In this study, blood thrombopoietin (TPO) levels were measured by ELISA in six TMD patients and the expression of c-Mpl, a ligand for TPO, was examined on the blast cells from four patients by flow cytometer. At the onset, TPO level was undetectable in one patient and significantly lower in five patients than six neonatal controls (mean 0.52 fmol/ml, range 0.30-0.93 vs. 3.70, 1.38-8.33, P < 0.001), although platelet counts were similar (mean 321 x 10(9)/l, range 42-1,040 vs. 253 x 10(9)/l, 124-381). Two patients died of hepatic failure. TPO levels were measured in five patients after regression of the blast cells. With regression of blast cells, TPO levels were remarkably increased in four survived patients. In one patient with hepatic failure, TPO level was poorly elevated and relatively lower compared to the others. TPO levels were inversely correlated with blast numbers (r = -0.85, P < 0.001), but not with platelet counts (r = 0.426). Blast cells from four patients were all positive for c-Mpl. Our findings suggest that megakaryocyte mass is a major regulator of TPO levels and hepatic failure may affect the TPO level because liver is a major source of TPO production.

Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPO-induced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPO-induced proliferation and differentiation, c-mpl was introduced into F-36P, a human interleukin-3 (IL-3)-dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate and differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F-36P-mpl cells, and their effects on TPO-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and dn STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by approximately 30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-rasG12V) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-rasG12V-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.

Thrombopoietin (TPO), or c-MPL ligand, is the primary regulator of megakaryocyte and platelet production. TPO receptors expressed on human megakaryocytes derived from peripheral blood (PB) and cord blood (CB) progenitors cultured in the presence of TPO have now been analyzed quantitatively. Like those on human PB platelets, TPO receptors on the cultured megakaryocytes exhibited a molecular mass of approximately 80 kDa. Various characteristics of PB- and CB-derived megakaryocytes indicated that the former were more mature than the latter. Both PB- and CB-derived megakaryocytes expressed a single class of high-affinity TPO receptors, with 1933 +/- 772 (n = 3) and 184 +/- 48 (n = 4) sites per cell, respectively. These data indicate that the number of TPO receptors on human megakaryocytes increases with cell maturation.

Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

Proto-oncogene c-mpl is structurally homologous with the hematopoietic growth factor receptor superfamily. The ligand for c-mpl was purified and its gene cloned. Extensive functional studies revealed that the ligand for c-mpl plays a crucial role in megakaryocytopoiesis and platelet production, hence, this ligand proved to be the long-sought hematopoietin, thrombopoietin (TPO). We briefly review here the role for TPO in early hematopoiesis, based on our in vitro data obtained using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) but not TPO alone supported the growth of murine primitive hematopoietic progenitors. Studies on lineage expression indicated that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of primitive hematopoietic progenitors. TPO also hastened IL-3-dependent growth of progenitors by shortening the time required for cell-cycling. While size of the colonies did not differ between colonies supported by IL-3 alone and those supported by IL-3 plus TPO, the incidence of megakaryocyte-containing colonies in cultures supported by IL-3 plus TPO was higher than that in cultures supported by IL-3 alone. Taken together, TPO as a single factor can support the survival of hematopoietic progenitors and TPO synergizes with SF or IL-3 to be active on early multipotential hematopoietic progenitors. These findings suggest that the function of TPO initially thought to be restricted to the megakaryocytic lineage proved to be greater in hematopoiesis. Reports from other laboratories regarding the involvement of TPO in early hematopoiesis are also discussed.

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis through binding to the cytokine receptor c-Mpl (the product of the c-mpl proto-oncogene). In an effort to determine the pathophysiological role of TPO-c-Mpl system in essential thrombocythemia (ET), we have examined the levels of serum TPO and the expression and function of platelet c-Mpl in 17 patients with ET. In spite of extreme thrombocytosis, serum TPO levels were slightly elevated or within normal range in most, if not all, patients with ET (mean +/- SD, 1.31 +/- 1.64 fmol/mL), as compared with normal subjects (0.76 +/- 0.21 fmol/mL). Flow cytometric and Western blot analyses revealed that the expression of platelet c-Mpl was strikingly reduced in all patients with ET. Furthermore, the expression of platelet c-mpl mRNA was found to be significantly decreased in the ET patients tested. In contrast, almost identical levels of GPIIb/IIIa protein and mRNA were expressed in platelets from ET patients and normal controls. In addition to expression level, activation state of platelet c-Mpl was investigated in ET patients. Immunoblotting with anti-phosphotyrosine antibody showed that no aberrant protein-tyrosine phosphorylation was observed in platelets of ET patients before treatment with TPO, and the levels of TPO-induced protein-tyrosine phosphorylation, including c-Mpl-tyrosyl phosphorylation, roughly paralleled those of c-Mpl expression, suggesting that c-Mpl-mediated signaling pathway was not constitutively activated in platelets of ET patients. These results suggested that the TPO-c-Mpl system may not be directly linked to pathogenesis of ET, and that gene(s) mutated in ET may be important in regulating the levels of c-mpl gene expression in addition to the growth and differentiation of multipotential hematopoietic stem cells.

To determine the hematopoietic actions of recombinant human c-Mpl ligand (thrombopoietin [TPO]), we studied its effects on the proliferation and differentiation of highly purified CD34+ blood progenitors in plasma-containing and serum-free culture. TPO alone promoted the growth of small megakaryocyte colonies (CFU-Meg) in numbers two to three times greater than those produced by interleukin (IL)-3. The combination of TPO and stem cell factor (SCF) exerted a significant synergistic effect on CFU-Meg formation. In the presence of TPO and IL-3 or granulocyte/macrophage-colony stimulating factor (GM-CSF), a significant number of mixed colonies (CFU-Mix) were observed. The combination of TPO and Epo did not increase the number of CFU-Meg, but did support erythroid-burst (BFU-E) and CFU-Mix colony formation. Interestingly, the combination of TPO with cytokines known to have burst-promoting activity (BPA), including IL-3, GM-CSF, IL-9, and SCF, increased the number of BFU-E and CFU-Mix in the presence of Epo. The BPA of TPO was further investigated by delayed addition of Epo on day 6 after incubation with TPO from day 0. None of the BFU-E or CFU-Mix survived, indicating that TPO acted as a costimulant exclusively for Epo. Moreover, a neutralizing anti-human Mpl receptor polyclonal antibody completely abrogated the BPA of TPO, demonstrating that this effect was mediated through the Mpl receptor. Finally, experiments in single-cell clone sorting and serum-free culture clearly demonstrated that a combination of TPO and Epo directly supported BFU-E and CFU-Mix. These results suggest that TPO acts not only in megakaryocytopoiesis but also in the early stage of hematopoiesis.

In an effort to establish the specificity of the thrombopoietin (TPO) effects on murine multipotential progenitors, we tested the effects of soluble TPO receptor (sTPOR; sMpl) on multilineage colony formation that was supported by a combination of TPO and steel factor (SF). Surprisingly, sTPOR did not suppress colony formation from primitive progenitors. This led to the discovery that sTPOR synergizes with SF or Flt3/Flk2 ligand (FL) to support the formation of various types of hematopoietic colonies including multilineage colonies. The colonies supported by the combination of sTPOR and SF were capable of expressing both myeloid and B-lymphoid potentials. Studies using micromanipulation and serum-free culture showed that the effects of sTPOR and SF on the primitive progenitors are direct, not mediated by contaminating stromal cells, and not dependent on factors present in the serum. TPOR belongs to the cytokine receptor group that includes granulocyte colony-stimulating factor receptor (G-CSFR) and erythropoietin receptor (EPOR). Therefore, we tested the effects of sG-CSFR and sEPOR on primitive progenitors. sG-CSFR, but not sEPOR, was able to synergize with SF or FL in supporting the proliferation of primitive progenitors. The direct effects of the soluble receptors appear to be mediated through interactions with their respective membrane-bound receptors expressed on the primitive hematopoietic progenitors.

To investigate the functional domains for signal transduction of human Mpl, we constructed a series of human c-mpl cDNAs with various deletions in the cytoplasmic domain, and then introduced each cDNA into murine IL-3-dependent myeloid leukemia FDC/P2 cells to establish stable transformants. We examined the growth and differentiation responses and tyrosine phosphorylation of the intracellular signaling proteins including Jak2, Tyk2, Stat3, Stat5, Vav, SHPTP2, Cbl, Shc and Shc-associated p145 when receptor stimulation occurred after thrombopoietin (TPO) binding. TPO stimulated cell proliferation and induced the expression of megakaryocyte lineage-specific AP-51 and CD61 cell surface antigens and tyrosine phosphorylation of the signaling proteins in transformants expressing full length human Mpl. These results suggested that Mpl not only induced proliferation but also transduced megakaryocyte-specific differentiation signals into FDC/P2 cells. Mutational analysis of human Mpl indicated that the N-terminal region of its cytoplasmic domain is necessary and sufficient to transduce proliferation and differentiation signals into cells, while the C-terminal region may also play important roles in transducing the differentiation signals.

Thrombopoietin (TPO) is implicated as a primary regulator of megakaryopoiesis and thrombopoiesis. However, the biologic effects of TPO on human acute myeloblastic leukemia (AML) cells are largely unknown. To determine if recombinant human (rh) TPO has proliferation-supporting and differentiation-inducing activities in AML cells, 15 cases of AML cells that were exclusively composed of undifferentiated leukemia cells and showed growth response to rhTPO in a short-term culture (72 hours) were subjected to long-term suspension culture with or without rhTPO. Of 15 cases, rhTPO supported proliferation of AML cells for 2 to 4 weeks in 4 cases whose French-American-British subtypes were M0, M2, M4, and M7, respectively. In addition to the proliferation-supporting activity, rhTPO was found to induce AML cells to progress to some degree of megakaryocytic differentiation at both morphologic and surface-phenotypic level in 2 AML cases with M0 and M7 subtypes. The treatment of AML cells with rhTPO resulted in rapid tyrosine phosphorylation of the TPO-receptor, c-mpl, and STAT3 in all of cases tested. By contrast, the expression of erythroid/megakaryocyte-specific transcription factors (GATA-1, GATA-2, and NF-E2) was markedly induced or enhanced in only 2 AML cases that showed megakaryocytic differentiation in response to rhTPO. These results suggested that, at least in a fraction of AML cases, TPO could not only support the proliferation of AML cells irrespective of AML subtypes, but could also induce megakaryocytic differentiation, possibly through activation of GATA-1, GATA-2, and NF-E2.

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c-Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c-Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.

Thrombopoietin (TPO) is a recently identified hematopoietic growth factor that is essential for the growth and development of megakaryocytes. We have previously shown that TPO induces proliferation of acute myeloblastic leukemia (AML) cells in vitro. In this study, we have examined the expression of TPO and its receptor c-mpl in a series of AML cases and human leukemia cell lines. The mRNA transcripts of TPO were detectable in 18 of 50 AML cases and in some myeloid leukemia cell lines (HEL, M07E and CMK) by means of reverse transcriptase polymerase chain reaction (RT-PCR). In addition, TPO transcripts were coexpressed with c-mpl transcripts in 10 of 50 AML cases and in HEL, M07E and CMK cells. With regard to the French-American-British (FAB) classification, coexpression OF TPO and c-mpl was observed with high frequency in AML cases of M7-type. Despite the TPO expression in a substantial fraction of leukemia cells, biological activity of TPO was not found in the conditioned medium that was obtained from cultivation of TPO mRNA-positive leukemia cells. These results suggest that TPO may not commonly participate in the abnormal growth of AML cells as an extracellular autocrine growth factor.

The leakage of heart-type cytoplasmic fatty acid-binding protein (H-FABPc) from injured myocardial cells has been reported. We have previously proposed that its plasma and urinary levels could be used as an early indicator of myocardial injury and also reflect the severity of myocardial injury. To confirm this hypothesis, the time course of changes of the plasma and urinary H-FABPc was investigated during myocardial injury induced by coronary artery occlusion and reperfusion in dogs. The plasma elimination kinetics and urinary excretion kinetics of H-FABPc were also analysed in dogs which were given a bolus injection of exogenous H-FABPc. The distribution of circulating H-FABPc was determined in mice by whole-body autoradiography using 125I-labelled H-FABPc. In myocardial injury model, plasma and urinary H-FABPc level showed rapid increase after reperfusion. The elimination kinetic study revealed that H-FABPc was mono-exponentially cleared from the circulation. The elimination rate constant (Ke) was 0.0275 +/- 0.0094/min (mean +/- S.D., n = 7) and the disappearance half-time (t1/2) was 27.5 +/- 8.4 min (mean +/- S.D., n = 7). Exogenous H-FABPc appeared in urine soon after administration, with the peak level being at 6.9 +/- 2.0 min (mean +/- S.D., n = 7). Whole-body autoradiography also demonstrated that 125I-H-FABPc accumulated rapidly in the kidneys. This study demonstrated that H-FABPc leaked rapidly from injured myocardium and rapidly appeared in plasma and urine. Infarct size was closely correlated with the calculated H-FABPc release (r = 0.89, r2 = 0.80, P < 0.01, n = 7) and with the amount of the urinary H-FABPc (r = 0.94, r2 = 0.88, P < 0.01, n = 7). These data suggest that measurement of the H-FABPc levels in plasma and urine might be useful for the early detection of myocardial injury and also for the assessment of infarct size.

The serum level of CA 19-9 elevated exponentially in gastrointestinal carcinoma and the specificity of CA 19-9 was extraordinarily high. The sensitivity of CA 19-9 to pancreas, bile duct, hepatocellular and metastatic liver carcinoma was 75%, 79%, 22%, and 82% each. TPA seems to be useful as a screening tumor maker of gastrointestinal carcinoma, because the sensitivity was very high (84%) in every gastrointestinal carcinoma. IAP increased in pancreas and bile duct carcinoma and reflected immunological status of tumor bearing host. 5'-NPD-V was useful tumor marker not only of hepatocellular carcinoma but also pancreas and bile duct carcinoma.