TGF-β-induced growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active p38 MAPK

<p>Abstract</p> <p>Background</p> <p>Cytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.</p> <p>Results</p> <p>TGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.</p> <p>Conclusions</p> <p>We suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.</p

Goats without prion protein display enhanced proinflammatory pulmonary signaling and extracellular matrix remodeling upon systemic lipopolysaccharide challenge

A naturally occurring mutation in the PRNP gene of Norwegian dairy goats terminates synthesis of the cellular prion protein (PrPC), rendering homozygous goats (PRNPTer/Ter) devoid of the protein. Although PrPC has been extensively studied, particularly in the central nervous system, the biological role of PrPC remains incompletely understood. Here, we examined whether loss of PrPC affects the initial stage of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Acute pulmonary inflammation was induced by intravenous injection of LPS (Escherichia coli O26:B6) in 16 goats (8 PRNPTer/Ter and 8 PRNP+/+). A control group of 10 goats (5 PRNPTer/Ter and 5 PRNP+/+) received sterile saline. Systemic LPS challenge induced sepsis-like clinical signs including tachypnea and respiratory distress. Microscopic examination of lungs revealed multifocal areas with alveolar hemorrhages, edema, neutrophil infiltration, and higher numbers of alveolar macrophages, with no significant differences between PRNP genotypes. A total of 432 (PRNP+/+) and 596 (PRNPTer/Ter) genes were differentially expressed compared with the saline control of the matching genotype. When assigned to gene ontology categories, biological processes involved in remodeling of the extracellular matrix (ECM), were exclusively enriched in PrPC-deficient goats. These genes included a range of collagen-encoding genes, and proteases such as matrix metalloproteinases (MMP1, MMP2, MMP14, ADAM15) and cathepsins. Several proinflammatory upstream regulators (TNF-α, interleukin-1β, IFN-γ) showed increased activation scores in goats devoid of PrPC. In conclusion, LPS challenge induced marked alterations in the lung tissue transcriptome that corresponded with histopathological and clinical findings in both genotypes. The increased activation of upstream inflammatory regulators and enrichment of ECM components could reflect increased inflammation in the absence of PrPC. Further studies are required to elucidate whether these alterations may affect the later reparative phase of ALI

Goats without Prion Protein Display Enhanced Proinflammatory Pulmonary Signaling and Extracellular Matrix Remodeling upon Systemic Lipopolysaccharide Challenge

A naturally occurring mutation in the PRNP gene of Norwegian dairy goats terminates synthesis of the cellular prion protein (PrPC), rendering homozygous goats (PRNPTer/Ter) devoid of the protein. Although PrPC has been extensively studied, particularly in the central nervous system, the biological role of PrPC remains incompletely understood. Here, we examined whether loss of PrPC affects the initial stage of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Acute pulmonary inflammation was induced by intravenous injection of LPS (Escherichia coli O26:B6) in 16 goats (8 PRNPTer/Ter and 8 PRNP+/+). A control group of 10 goats (5 PRNPTer/Ter and 5 PRNP+/+) received sterile saline. Systemic LPS challenge induced sepsis-like clinical signs including tachypnea and respiratory distress. Microscopic examination of lungs revealed multifocal areas with alveolar hemorrhages, edema, neutrophil infiltration, and higher numbers of alveolar macrophages, with no significant differences between PRNP genotypes. A total of 432 (PRNP+/+) and 596 (PRNPTer/Ter) genes were differentially expressed compared with the saline control of the matching genotype. When assigned to gene ontology categories, biological processes involved in remodeling of the extracellular matrix (ECM), were exclusively enriched in PrPC-deficient goats. These genes included a range of collagen-encoding genes, and proteases such as matrix metalloproteinases (MMP1, MMP2, MMP14, ADAM15) and cathepsins. Several proinflammatory upstream regulators (TNF-α, interleukin-1β, IFN-γ) showed increased activation scores in goats devoid of PrPC. In conclusion, LPS challenge induced marked alterations in the lung tissue transcriptome that corresponded with histopathological and clinical findings in both genotypes. The increased activation of upstream inflammatory regulators and enrichment of ECM components could reflect increased inflammation in the absence of PrPC. Further studies are required to elucidate whether these alterations may affect the later reparative phase of ALI

Loss of prion protein induces a primed state of type I interferon-responsive genes

<div><p>The cellular prion protein (PrP^C) has been extensively studied because of its pivotal role in prion diseases; however, its functions remain incompletely understood. A unique line of goats has been identified that carries a nonsense mutation that abolishes synthesis of PrP^C. In these animals, the PrP-encoding mRNA is rapidly degraded. Goats without PrP^C are valuable in re-addressing loss-of-function phenotypes observed in <i>Prnp</i> knockout mice. As PrP^C has been ascribed various roles in immune cells, we analyzed transcriptomic responses to loss of PrP^C in peripheral blood mononuclear cells (PBMCs) from normal goat kids (<i>n</i> = 8, <i>PRNP</i>^+/+) and goat kids without PrP^C (<i>n</i> = 8, <i>PRNP</i>^Ter/Ter) by mRNA sequencing. PBMCs normally express moderate levels of PrP^C. The vast majority of genes were similarly expressed in the two groups. However, a curated list of 86 differentially expressed genes delineated the two genotypes. About 70% of these were classified as interferon-responsive genes. In goats without PrP^C, the majority of type I interferon-responsive genes were in a primed, modestly upregulated state, with fold changes ranging from 1.4 to 3.7. Among these were <i>ISG15</i>, <i>DDX58</i> (RIG-1), <i>MX1</i>, <i>MX2</i>, <i>OAS1</i>, <i>OAS2 and DRAM1</i>, all of which have important roles in pathogen defense, cell proliferation, apoptosis, immunomodulation and DNA damage response. Our data suggest that PrP^C contributes to the fine-tuning of resting state PBMCs expression level of type I interferon-responsive genes. The molecular mechanism by which this is achieved will be an important topic for further research into PrP^C physiology.</p></div

Role of Smad Proteins in Resistance to BMP-Induced Growth Inhibition in B-Cell Lymphoma

<div><p>Bone morphogenetic protein (BMP) expression and signaling are altered in a variety of cancers, but the functional impact of these alterations is uncertain. In this study we investigated the impact of expression of multiple BMPs and their signaling pathway components in human B-cell lymphoma. <em>BMP</em> messages, in particular <em>BMP7</em>, were detected in normal and malignant B cells. Addition of exogenous BMPs inhibited DNA synthesis in most lymphoma cell lines examined, but some cell lines were resistant. Tumor specimens from three out of five lymphoma patients were also resistant to BMPs, as determined by no activation of the BMP effectors Smad1/5/8. We have previously shown that BMP-7 potently induced apoptosis in normal B cells, which was in contrast to no or little inhibitory effect of this BMP in the lymphoma cells tested. BMP-resistance mechanisms were investigated by comparing sensitive and resistant cell lines. While BMP receptors are downregulated in many cancers, we documented similar receptor levels in resistant and sensitive lymphoma cells. We found a positive correlation between activation of Smad1/5/8 and inhibition of DNA synthesis. Gene expression analysis of two independent data sets showed that the levels of inhibitory Smads varied across different B-cell lymphoma. Furthermore, stable overexpression of Smad7 in two different BMP-sensitive cell lines with low endogenous levels of <em>SMAD7</em>, rendered them completely resistant to BMPs. This work highlights the role of Smads in determining the sensitivity to BMPs and shows that upregulation of Smad7 in cancer cells is sufficient to escape the negative effects of BMPs.</p> </div

Expression of interferon-responsive genes in blood leukocytes after <i>in vivo</i> lipopolysaccharide (LPS) challenge in goats without PrP^C.

<p>RNA was extracted from circulating blood leukocytes from both genotypes, and gene expression was analyzed by FLUIDIGM qPCR. (A) Basal expression level (0 h) of selected interferon-responsive genes and <i>STAT1</i> in <i>PRNP</i>^+/+ (<i>n</i> = 12) and <i>PRNP</i>^Ter/Ter (<i>n</i> = 13) animals. (B) Gene expression of interferon-responsive genes and <i>STAT1</i> after <i>in vivo</i> LPS challenge (1 h) from <i>PRNP</i>^+/+ (<i>n</i> = 7) and <i>PRNP</i>^Ter/Ter (<i>n</i> = 8) animals. Values are mean ± SEM. Statistical significance is indicated by *, p-value < 0.05, as assessed by multiple t-tests.</p