Thrombocytopenia in c-mpl-deficient mice.

Thrombopoietin (TPO) is a cytokine that is involved in the regulation of platelet production. The receptor for TPO is c-Mpl. To further investigate the role and specificity of this receptor in regulating megakaryocytopoiesis, c-mpl-deficient mice were generated by gene targeting. The c-mpl-/- mice had an 85 percent decrease in their number of platelets and megakaryocytes but had normal amounts of other hematopoietic cell types. These mice also had increased concentrations of circulating TPO. These results show that c-mpl specifically regulates megakaryocytopoiesis and thrombopoiesis through activation by its ligand TPO.

Identification of synergistic signals initiating inner ear development.

Tissue manipulation experiments in amphibians more than 50 years ago showed that induction of the inner ear requires two signals: a mesodermal signal followed by a neural signal. However, the molecules mediating this process have remained elusive. We present evidence for mesodermal initiation of otic development in higher vertebrates and show that the mesoderm can direct terminal differentiation of the inner ear in rostral ectoderm. Furthermore, we demonstrate the synergistic interactions of the extracellular polypeptide ligands FGF-19 and Wnt-8c as mediators of mesodermal and neural signals, respectively, initiating inner ear development.

Identification of a new member of the tumor necrosis factor family and its receptor, a human ortholog of mouse GITR.

The tumor necrosis factor (TNF) and TNF receptor (TNFR) gene superfamilies regulate diverse biological functions, including cell proliferation, differentiation, and survival [1] [2] [3]. We have identified a new TNF-related ligand, designated human GITR ligand (hGITRL), and its human receptor (hGITR), an ortholog of the recently discovered murine glucocorticoid-induced TNFR-related (mGITR) protein [4]. The hGITRL gene mapped to chromosome 1q23, near the gene for the TNF homolog Fas/CD95 ligand [5]. The hGITR gene mapped to chromosome 1p36, near a cluster of five genes encoding TNFR homologs [1] [6]. We found hGITRL mRNA in several peripheral tissues, and detected hGITRL protein on cultured vascular endothelial cells. The levels of hGITR mRNA in tissues were generally low; in peripheral blood T cells, however, antigen-receptor stimulation led to a substantial induction of hGITR transcripts. Cotransfection of hGITRL and hGITR in embryonic kidney 293 cells activated the anti-apoptotic transcription factor NF-kappaB, via a pathway that appeared to involve TNFR-associated factor 2 (TRAF2) [7] and NF-kappaB-inducing kinase (NIK) [8]. Cotransfection of hGITRL and hGITR in Jurkat T leukemia cells inhibited antigen-receptor-induced cell death. Thus, hGITRL and hGITR may modulate T lymphocyte survival in peripheral tissues.

The role of the CCAAT/enhancer-binding protein in the transcriptional regulation of the gene for phosphoenolpyruvate carboxykinase (GTP).

Previous studies have identified a region in the promoter of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (positions -460 to +73) containing the regulatory elements which respond to cyclic AMP, glucocorticoids, and insulin and confer the tissue- and developmental stage-specific properties to the gene. We report that CCAAT/enhancer-binding protein (C/EBP) binds to the cyclic AMP-responsive element CRE-1 as well as to two regions which have been previously shown to bind proteins enriched in liver nuclei. The DNase I footprint pattern provided by the recombinant C/EBP was identical to that produced by a 43-kDa protein purified from rat liver nuclear extracts, using a CRE oligonucleotide affinity column, which was originally thought to be the CRE-binding protein CREB. Transient contransfection experiments using a C/EBP expression vector demonstrated that C/EBP could trans activate the PEPCK promoter. The trans activation occurred through both the upstream, liver-specific protein-binding domains and the CRE. The CRE-binding protein bound only to CRE-1 and not to the upstream C/EBP-binding sites. The results of this study, along with physiological properties of C/EBP, indicate an important role for this transcription factor in providing the PEPCK gene with several of its regulatory characteristics.

Antisense transcripts and protein binding motifs within the Wilms tumour (WT1) locus.

Transcription of the WT1 locus is restricted, both temporally and spatially, to a subset of epithelial cells in mammalian kidneys and gonads. WT1, one of the two divergent transcripts mapping to this locus encodes a zinc finger protein that is likely a transcriptional regulator. The other transcript, WIT1, encodes a product of unknown function that is subject to alternate splicing in the region immediately 5' of the WT1 gene. Analysis of the 5' end of this locus further revealed the presence of multiple transcriptional start sites for both genes, such that some of the WIT1 transcripts are encoded by the antisense strand of the first exon of WT1. The genomic region surrounding the transcriptional start sites appears to constitute part of a bi-directional promoter based on the ability of a DNA fragment derived from this region to direct expression of a chimeric CAT gene construct in transient transfection assays. Discrete sequences within the region are capable of interaction in vitro with nuclear extracts derived from a variety of rat and mouse tissues. Interestingly, recombinant WT1, representing the product of zinc finger region of the most abundant of the four alternatively spliced transcripts, is also capable of binding to sequences within this region.

Modulation of hormone response elements by promoter environment.

Hormone response elements (HREs) are nucleotide sequences that confer onto promoters the ability to alter their transcriptional pace in response to hormones. Growing evidence indicates that the functional activity of HREs can be significantly modulated by their promoter environment, making it possible for genes containing the same HRE to display diversity in their responsiveness to a given hormone signal.

Acinar cells of the pancreas are a target of interleukin-22.

Interleukin-22 (IL-22) (also reported as IL-10-related T cell-derived inducible factor, IL-TIF) is a recently identified cytokine found to signal through a receptor comprising the class II cytokine receptor family members IL-10Rbeta/CRF2-4 and IL-22R. Previous work has established that IL-10Rbeta, also a component of the IL10R complex, exhibits a broad distribution of mRNA expression. Here, we observe that IL-22R exhibits a restricted expression pattern, with highest levels of mRNA expression in pancreas and detectable expression in multiple other tissues, particularly liver, small intestine, colon, and kidney. We find that isolated primary pancreatic acinar cells and the acinar cell line 266-6 respond to IL-22 with activation of Stat3 and changes in gene transcription. IL-22 mediates robust induction of mRNA for pancreatitis-associated protein (PAP1)/Reg2 and osteopontin (OPN). PAP1 is a secreted protein related to the Reg family of trophic factors and was initially characterized as a protein elevated in pancreatitis. In vivo injection of IL-22 resulted in rapid induction of PAP1 in pancreas, a response not observed in mice deficient in IL-10Rbeta. These results support the conclusion that IL-10Rbeta is a required common component of both the IL-10 and IL-22 receptors and suggest that IL-22 may play a role in the immune response in pancreas.

Infection time and density influence the response of sorghum to the parasitic angiosperm Striga hermonthica.

Two cultivars of sorghum (CSH-1 and Ochuti) were grown in the presence and absence of the root hemiparasite Striga hermonthica in uniform conditions in the field in Kenya, Africa. S. hermonthica had a marked influence on growth and photosynthesis of 'CSH-1'; however, 'Ochuti' showed a less severe response to infection and tolerance of the parasite. The variation in genotype response might be partly explained by later attachment of the parasite and a lower level of infection. Laboratory studies were used to determine the importance of both variables in determining host response to infection. Early infection by S. hermonthica had a more negative effect on the host than late infection. The level of parasite biomass supported by the host also influenced host productivity but the relationship was nonlinear. Low degrees of parasite infection had a proportionately much greater effect on host grain weight than at greater parasite loading. Early infection of 'Ochuti' in laboratory conditions resulted in lower stem dry weight than in uninfected plants but not in smaller total plant biomass or lower rates of photosynthesis. In conclusion, the time of parasite attachment affected host performance and might explain much of the variation in host sensitivity both within and between studies. The level of parasite infection affected host performance to a lesser extent. In addition, late attachment and low levels of infection might have implications for control management strategies.

Novel sources of resistance to Striga hermonthica in Tripsacum dactyloides, a wild relative of maize.

• The parasitic weed Striga hermonthica lowers cereal yield in small-holder farms in Africa. Complete resistance in maize to S. hermonthica infection has not been identified. A valuable source of resistance to S. hermonthica may lie in the genetic potential of wild germplasm. • The susceptibility of a wild relative of maize, Tripsacum dactyloides and a Zea mays-T. dactyloides hybrid to S. hermonthica infection was determined. Striga hermonthica development was arrested after attachment to T. dactyloides. Vascular continuity was established between parasite and host but there was poor primary haustorial tissue differentiation on T. dactyloides compared with Z. mays. Partial resistance was inherited in the hybrid. • Striga hermonthica attached to Z. mays was manipulated such that different secondary haustoria could attach to different hosts. Secondary haustoria formation was inhibited on T. dactyloides, moreover, subsequent haustoria formation on Z. mays was also impaired. • Results suggest that T. dactyloides produces a signal that inhibits haustorial development: this signal may be mobile within the parasite haustorial root system.

Plant eats plant: sap-feeding witchweeds and other parasitic angiosperms.

About one in every hundred species of flowering plant is parasitic and obtain some or all of their carbon, nutrients and water from the sap of their hosts. They possess unique morphological and metabolic adaptations but are more than just botanical curiosities.

A novel form of resistance in rice to the angiosperm parasite Striga hermonthica.

The root hemiparasitic weed Striga hermonthica is a serious constraint to grain production of economically important cereals in sub-Saharan Africa. Breeding for parasite resistance in cereals is widely recognized as the most sustainable form of long-term control; however, advances have been limited owing to a lack of cereal germplasm demonstrating postattachment resistance to Striga. Here, we identify a cultivar of rice (Nipponbare) that exhibits strong postattachment resistance to S. hermonthica; the parasite penetrates the host root cortex but does not form parasite-host xylem-xylem connections. In order to identify the genomic regions contributing to this resistance, a mapping population of backcross inbred lines between the resistant (Nipponbare) and susceptible (Kasalath) parents were evaluated for resistance to S. hermonthica. Composite interval mapping located seven putative quantitative trait loci (QTL) explaining 31% of the overall phenotypic variance; a second, independent, screen confirmed four of these QTL. Relative to the parental lines, allelic substitutions at these QTL altered the phenotype by at least 0.5 of a phenotypic standard deviation. Thus, they should be regarded as major genes and are likely to be useful in breeding programmes to enhance host resistance.

Dissection of c-Mpl and thrombopoietin function: studies of knockout mice and receptor signal transduction.

The physiological roles and mechanisms of action of thrombopoietin (TPO) and its receptor c-Mpl have been studied through the analysis of mice genetically deficient in these molecules, as well as through the dissection of signaling events utilizing chimeric receptors. The evidence clearly demonstrates that the TPO/c-Mpl system provides dominant control in the regulation of megakaryocytopoiesis. The signaling mechanisms that underlie this process appear to be similar to those noted with other members of the hematopoietic cytokine and cytokine receptor families.

Opposing actions of Fos and Jun on transcription of the phosphoenolpyruvate carboxykinase (GTP) gene. Dominant negative regulation by Fos.

Jun homodimers and Fos/Jun heterodimers bind to the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) at three sites within the first 350 base pairs of the promoter. These include CRE-1 (-82 to -90), and P3(II) and P4 (-252 to -258 and -268 to -285, respectively). Over-expression of Jun in HepG2 cells resulted in a 10-15-fold increase in the level of transcription of a chimeric PEPCK (-490 to +73)-CAT gene, while expression of Fos decreased transcription and blocked the induction of transcription from the PEPCK promoter by Jun. The action of Fos and Jun on PEPCK gene transcription involved each of the Fos/Jun-binding sites and was modulated by additional transcriptional regulatory elements within the PEPCK promoter. The ability of Fos to inhibit PEPCK transcription was dependent upon P3(I), a region of the promoter which does not bind Fos/Jun heterodimers, but does bind members of the C/EBP family of transcription factors. Stimulation of PEPCK transcription by 8-Br-cAMP or by overexpression of the catalytic subunit of protein kinase A was inhibited by Fos expression. The inhibitory effects of phorbol esters and protein kinase C on PEPCK gene expression may be mediated through the action of Fos and Jun.

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.

Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.

Relative roles of CCAAT/enhancer-binding protein beta and cAMP regulatory element-binding protein in controlling transcription of the gene for phosphoenolpyruvate carboxykinase (GTP).

The gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is expressed in a tissue-specific manner in the liver, kidney, and adipose tissue and is regulated by hormones including cAMP and insulin. Previous studies have shown that the CCAAT/enhancer-binding protein alpha (C/EBP alpha) binds to several sites on the PEPCK promoter and activates transcription from the promoter in hepatoma cells. Here, we report that a second member of the C/EBP family, C/EBP beta, bound to the same sites on the PEPCK promoter. However, C/EBP beta stimulated transcription primarily through the cAMP-responsive element (CRE), which maps between positions -77 to -94, but not at the more 5'-binding sites. In addition, the nuclear factor-1 site, which is immediately adjacent to the CRE in the PEPCK promoter, was also required for the full response of the promoter to cotransfected C/EBP beta. In gel mobility assays, antibodies to both C/EBP beta and the cAMP regulatory element-binding protein (CREB), but not to C/EBP alpha, "supershifted" DNA-protein complexes formed between a synthetic CRE oligomer and proteins prepared from rat liver nuclei. C/EBP beta mRNA was expressed at low levels in both the periportal and pericentral regions of the liver lobule, whereas expression of the gene for C/EBP alpha was confined to the pericentral region of the liver lobule. PEPCK gene transcription is greatest in the periportal region of the liver. CREB also bound to the CRE and stimulated transcription of a PEPCK-CAT vector in the presence of an expression vector for the catalytic subunit of protein kinase A. C/EBP beta and CREB bound to the CRE with similar affinities, both of which were greater than the affinity of C/EBP alpha. Within 90 min after the administration of dibutyryl cAMP to rats, there was a marked increase in the hepatic concentration of C/EBP beta mRNA and a decrease in the level of mRNA for C/EBP alpha. These studies indicate that C/EBP beta can regulate PEPCK gene transcription by acting through the CRE and that C/EBP beta, together with CREB, may contribute to the cAMP responsiveness of the PEPCK promoter.

The promoter regulatory regions of the genes for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken and the rat have different species-specific roles in gluconeogenesis.

Hepatic expression of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5' of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein alpha, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein alpha or CAAT/enhancer binding protein beta markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between -210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.

Cyclic AMP induction of phosphoenolpyruvate carboxykinase (GTP) gene transcription is mediated by multiple promoter elements.

The cis elements involved in the cAMP regulation of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) were studied by introducing a series of block mutations (10-15 base pairs of random sequence) into eight of the protein binding domains in a region of the promoter between -490 and +73. Each mutant promoter was ligated to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected into HepG2 cells. Transcription of PEPCK-CAT was stimulated 4-fold by the addition of 8-bromo-cAMP (8-Br-cAMP), whereas overexpression of the catalytic subunit of protein kinase A in these cells increased transcription from the PEPCK promoter 30-fold. Several elements within the PEPCK promoter acted synergistically to mediate this effect. These include CRE-1 (-92 to -82) and a complex unit from -220 to -280 composed of multiple binding sites termed P3(I) (-250 to -234), P3(II) (-260 to -250), and P4 (-286 to -270). Mutation of both CRE-1 and P3(I) resulted in the complete elimination of transcriptional induction by either 8-Br-cAMP or the catalytic subunit of protein kinase A. To examine the proteins involved in this response, we replaced CRE-1, which binds both C/EBP and cAMP-responsive element binding protein (CREB), with an optimal C/EBP binding sequence which significantly decreased the binding of CREB, but maintained the affinity for C/EBP. Transcription from this modified promoter was induced by 8-Br-cAMP and the catalytic subunit of protein kinase A (PKA) to a similar extent as noted with the native PEPCK promoter. However, the results of experiments involving cotransfection of PEPCK-CAT with expression vectors for PKA and either C/EBP or CREB suggest that CREB is capable of mediating a greater responsiveness to PKA than C/EBP. Our results indicate that multiple cis elements are involved in the cAMP induction of PEPCK gene transcription and that C/EBP and CREB are potentially involved in this response.

Identification of a thyroid hormone response element in the phosphoenolpyruvate carboxykinase (GTP) gene. Evidence for synergistic interaction between thyroid hormone and cAMP cis-regulatory elements.

Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in the liver is regulated by many hormones including thyroid hormone (T3). In order to identify the elements in the promoter which are required for transcriptional induction by T3, we cotransfected a T3 receptor expression vector with a PEPCK-CAT reporter gene into HepG2 cells. Using vectors with deletions in the PEPCK promoter, we identified a single T3 response element (TRE) between positions -332 and -308. This element binds [125I]T3-labeled T3 receptor contained in nuclear extracts prepared from rat liver. Furthermore, the P3(I) element (-250 to -234), a previously described cis-sequence involved in mediating the induction of PEPCK gene transcription by cAMP, is also required for the T3 responsiveness of the promoter. In the absence of either the TRE or the P3(I) binding sites, no stimulation of transcription from the PEPCK promoter by T3 was observed, indicating that both elements are required for the T3 transcriptional regulation. Finally, a synergistic induction of PEPCK gene transcription by T3 and cAMP is described. This interaction requires both T3- and cAMP-responsive cis-acting elements.

Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin.

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q27-28.