Stress Resilience of Spermatozoa and Blood Mononuclear Cells without Prion Protein.

The cellular prion protein PrPC is highly expressed in neurons, but also present in non-neuronal tissues, including the testicles and spermatozoa. Most immune cells and their bone marrow precursors also express PrPC. Clearly, this protein operates in highly diverse cellular contexts. Investigations into putative stress-protective roles for PrPC have resulted in an array of functions, such as inhibition of apoptosis, stimulation of anti-oxidant enzymes, scavenging roles, and a role in nuclear DNA repair. We have studied stress resilience of spermatozoa and peripheral blood mononuclear cells (PBMCs) derived from non-transgenic goats that lack PrPC (PRNPTer/Ter) compared with cells from normal (PRNP+/+) goats. Spermatozoa were analyzed for freeze tolerance, DNA integrity, viability, motility, ATP levels, and acrosome intactness at rest and after acute stress, induced by Cu2+ ions, as well as levels of reactive oxygen species (ROS) after exposure to FeSO4 and H2O2. Surprisingly, PrPC-negative spermatozoa reacted similarly to normal spermatozoa in all read-outs. Moreover, in vitro exposure of PBMCs to Doxorubicin, H2O2 and methyl methanesulfonate (MMS), revealed no effect of PrPC on cellular survival or global accumulation of DNA damage. Similar results were obtained with human neuroblastoma (SH-SY5Y) cell lines stably expressing varying levels of PrPC. RNA sequencing of PBMCs (n = 8 of PRNP+/+ and PRNPTer/Ter) showed that basal level expression of genes encoding DNA repair enzymes, ROS scavenging, and antioxidant enzymes were unaffected by the absence of PrPC. Data presented here questions the in vitro cytoprotective roles previously attributed to PrPC, although not excluding such functions in other cell types or tissues during inflammatory stress.

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-1ß, 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.

The cellular prion protein (PrPC) 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 PrPC. In these animals, the PrP-encoding mRNA is rapidly degraded. Goats without PrPC are valuable in re-addressing loss-of-function phenotypes observed in Prnp knockout mice. As PrPC has been ascribed various roles in immune cells, we analyzed transcriptomic responses to loss of PrPC in peripheral blood mononuclear cells (PBMCs) from normal goat kids (n = 8, PRNP+/+) and goat kids without PrPC (n = 8, PRNPTer/Ter) by mRNA sequencing. PBMCs normally express moderate levels of PrPC. 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 PrPC, 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 ISG15, DDX58 (RIG-1), MX1, MX2, OAS1, OAS2 and DRAM1, all of which have important roles in pathogen defense, cell proliferation, apoptosis, immunomodulation and DNA damage response. Our data suggest that PrPC 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 PrPC physiology.

The Cellular Prion Protein: A Player in Immunological Quiescence.

Despite intensive studies since the 1990s, the physiological role of the cellular prion protein (PrP(C)) remains elusive. Here, we present a novel concept suggesting that PrP(C) contributes to immunological quiescence in addition to cell protection. PrP(C) is highly expressed in diverse organs that by multiple means are particularly protected from inflammation, such as the brain, eye, placenta, pregnant uterus, and testes, while at the same time it is expressed in most cells of the lymphoreticular system. In this paradigm, PrP(C) serves two principal roles: to modulate the inflammatory potential of immune cells and to protect vulnerable parenchymal cells against noxious insults generated through inflammation. Here, we review studies of PrP(C) physiology in view of this concept.

Hematological shift in goat kids naturally devoid of prion protein.

The physiological role of the cellular prion protein (PrP(C)) is incompletely understood. The expression of PrP(C) in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrP(C) knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrP(C). Here we report hematological and immunological analyses of homozygous goat kids lacking PrP(C) (PRNP(Ter/Ter) ) compared to heterozygous (PRNP (+/Ter)) and normal (PRNP (+/+)) kids. Levels of cell surface PrP(C) and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNP (Ter/Ter), intermediate in PRNP (+/Ter) and high in PRNP (+/+) kids. The PRNP (Ter/Ter) animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrP(C) has a role in bone marrow physiology and warrant further studies of PrP(C) in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrP(C)-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrP(C).

Role of Smad proteins in resistance to BMP-induced growth inhibition in B-cell lymphoma.

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. BMP messages, in particular BMP7, 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 SMAD7, 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.

SARA is dispensable for functional TGF-ß signaling.

Smad anchor for receptor activation (SARA or ZFYVE9) has been proposed to mediate transforming growth factor ß (TGF-ß) signaling by direct interaction with the non-activated Smad proteins and the TGF-ß receptors; however, these findings are controversial. We demonstrate no correlation between SARA expression and the levels of TGF-ß-induced phosphorylation of Smads in various B-cell lymphomas. Moreover, knockdown of SARA in HeLa cells did not interfere with TGF-ß-induced Smad activation, Smad nuclear translocation, or induction of TGF-ß target genes. Various R-Smads and TGF-ß receptors did not co-immunoprecipitate with SARA. Collectively, our results demonstrate that SARA is dispensable for functional TGF-ß-mediated signaling.

Bone morphogenetic proteins inhibit CD40L/IL-21-induced Ig production in human B cells: differential effects of BMP-6 and BMP-7.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily. TGF-ß can affect class switch recombination in human B cells, but whether BMPs also play a role have not been tested. We investigated the functional effects of exogenously added BMPs on CD27(-) naive and CD27(+) memory B cells from healthy donors. BMP-2, -4, -6 and -7 inhibited CD40L/IL-21-induced production of IgM, IgG and IgA. BMP-6 reduced Ig production by 70% in memory B cells and more than 55% in naive B cells, whereas the other BMPs were slightly less potent. We observed a striking difference in functional effects between the structurally similar BMP-6 and BMP-7, as BMP-6 mainly inhibited plasmablast differentiation, and BMP-7 mainly induced apoptosis. In memory B cells, BMP-6 upregulated expression of DNA-binding protein inhibitor genes, but potently inhibited CD40L/IL-21-induced upregulation of the transcription factor XBP1, necessary for the late stages of plasmacytic differentiation. Expression of transcription factors regulating earlier stages (IRF4, PRDM1) was not affected by BMP-6. Taken together, these results show that BMPs are potent suppressors of naive and memory B cells.

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

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