PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway.

Little is known about eukaryotic chemorepulsion. The enzymes Phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3, and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion.

Differences between human male and female neutrophils in mRNA, translation efficiency, protein, and phosphoprotein profiles.

Background: Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive arthritis, and differences in neutrophil physiological responses such as a faster response to the chemorepellent SLIGKV. Little is known about the basis of sex-based differences in human neutrophils. Methods: Starting with human neutrophils from healthy donors, we used RNA-seq to examine total mRNA profiles, mRNAs not associated with ribosomes and thus not being translated, mRNAs in monosomes, and mRNAs in polysomes and thus heavily translated. We used mass spectrometry systems to identify proteins and phosphoproteins. Results: There were sex-based differences in the translation of 24 mRNAs. There were 132 proteins with higher levels in male neutrophils; these tended to be associated with RNA regulation, ribosome, and phosphoinositide signaling pathways, whereas 30 proteins with higher levels in female neutrophils were associated with metabolic processes, proteosomes, and phosphatase regulatory proteins. Male neutrophils had increased phosphorylation of 32 proteins. After exposure to SLIGKV, male neutrophils showed a faster response in terms of protein phosphorylation compared to female neutrophils. Conclusions: Male neutrophils have higher levels of proteins and higher phosphorylation of proteins associated with RNA processing and signaling pathways, while female neutrophils have higher levels of proteins associated with metabolism and proteolytic pathways. This suggests that male neutrophils might be more ready to adapt to a new environment, and female neutrophils might be more effective at responding to pathogens. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.

PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway.

The directed movement of eukaryotic cells is crucial for processes such as embryogenesis and immune cell trafficking. The enzyme Phosphatase and tensin homolog (PTEN) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. Dictyostelium discoideum cells require both PTEN and the PTEN-like phosphatase CnrN to locally inhibit Ras activation to induce biased movement of cells away from the secreted chemorepellent protein AprA. Both PTEN and CnrN decrease basal levels of PI(3,4,5)P 3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P 2 levels, decrease PI(3,4,5)P 3 levels, inhibit proliferation, decrease myosin II phosphorylation, and increase filopod sizes. AprA causes PTEN, similar to CnrN, to localize to the side of the cell towards AprA in an AprA gradient. However, PTEN and CnrN also have distinct roles in some signaling pathways. PTEN, but not CnrN, decreases basal levels of PI(4,5)P 2 , AprA requires PTEN, but not CnrN, to induce cell roundness, and CnrN and PTEN have different effects on the number of filopods and pseudopods, and the sizes of filopods. Together, our results suggest that CnrN and PTEN play unique roles in D. discoideum signaling pathways, and possibly dephosphorylate PI(3,4,5)P 3 in different membrane domains, to mediate chemorepulsion away from AprA.

A phosphatidylinositol phosphate kinase inhibits Ras activation and regulates chemorepulsion in Dictyostelium discoideum.

During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.

Sex-Based Differences in Human Neutrophil Chemorepulsion.

A considerable amount is known about how eukaryotic cells move toward an attractant, and the mechanisms are conserved from Dictyostelium discoideum to human neutrophils. Relatively little is known about chemorepulsion, where cells move away from a repellent signal. We previously identified pathways mediating chemorepulsion in Dictyostelium, and here we show that these pathways, including Ras, Rac, protein kinase C, PTEN, and ERK1 and 2, are required for human neutrophil chemorepulsion, and, as with Dictyostelium chemorepulsion, PI3K and phospholipase C are not necessary, suggesting that eukaryotic chemorepulsion mechanisms are conserved. Surprisingly, there were differences between male and female neutrophils. Inhibition of Rho-associated kinases or Cdc42 caused male neutrophils to be more repelled by a chemorepellent and female neutrophils to be attracted to the chemorepellent. In the presence of a chemorepellent, compared with male neutrophils, female neutrophils showed a reduced percentage of repelled neutrophils, greater persistence of movement, more adhesion, less accumulation of PI(3,4,5)P3, and less polymerization of actin. Five proteins associated with chemorepulsion pathways are differentially abundant, with three of the five showing sex dimorphism in protein localization in unstimulated male and female neutrophils. Together, this indicates a fundamental difference in a motility mechanism in the innate immune system in men and women.

Using Dictyostelium to Develop Therapeutics for Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) involves damage to lungs causing an influx of neutrophils from the blood into the lung airspaces, and the neutrophils causing further damage, which attracts more neutrophils in a vicious cycle. There are ∼190,000 cases of ARDS per year in the US, and because of the lack of therapeutics, the mortality rate is ∼40%. Repelling neutrophils out of the lung airspaces, or simply preventing neutrophil entry, is a potential therapeutic. In this minireview, we discuss how our lab noticed that a protein called AprA secreted by growing Dictyostelium cells functions as a repellent for Dictyostelium cells, causing cells to move away from a source of AprA. We then found that AprA has structural similarity to a human secreted protein called dipeptidyl peptidase IV (DPPIV), and that DPPIV is a repellent for human neutrophils. In animal models of ARDS, inhalation of DPPIV or DPPIV mimetics blocks neutrophil influx into the lungs. To move DPPIV or DPPIV mimetics into the clinic, we need to know how this repulsion works to understand possible drug interactions and side effects. Combining biochemistry and genetics in Dictyostelium to elucidate the AprA signal transduction pathway, followed by drug studies in human neutrophils to determine similarities and differences between neutrophil and Dictyostelium chemorepulsion, will hopefully lead to the safe use of DPPIV or DPPIV mimetics in the clinic.

Extracellular signaling in Dictyostelium.

In the last few decades, we have learned a considerable amount about how eukaryotic cells communicate with each other, and what it is the cells are telling each other. The simplicity of Dictyostelium discoideum, and the wide variety of available tools to study this organism, makes it the equivalent of a hydrogen atom for cell and developmental biology. Studies using Dictyostelium have pioneered a good deal of our understanding of eukaryotic cell communication. In this review, we will present a brief overview of how Dictyostelium cells use extracellular signals to attract each other, repel each other, sense their local cell density, sense whether the nearby cells are starving or stressed, count themselves to organize the formation of structures containing a regulated number of cells, sense the volume they are in, and organize their multicellular development. Although we are probably just beginning to learn what the cells are telling each other, the elucidation of Dictyostelium extracellular signals has already led to the development of possible therapeutics for human diseases.

An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells.

Eukaryotic chemoattraction signal transduction pathways, such as those used by Dictyostelium discoideum to move toward cAMP, use a G protein-coupled receptor to activate multiple conserved pathways such as PI3 kinase/Akt/PKB to induce actin polymerization and pseudopod formation at the front of a cell, and PTEN to localize myosin II to the rear of a cell. Relatively little is known about chemorepulsion. We previously found that AprA is a chemorepellent protein secreted by Dictyostelium cells. Here we used 29 cell lines with disruptions of cAMP and/or AprA signal transduction pathway components, and delineated the AprA chemorepulsion pathway. We find that AprA uses a subset of chemoattraction signal transduction pathways including Ras, protein kinase A, target of rapamycin (TOR), phospholipase A, and ERK1, but does not require the PI3 kinase/Akt/PKB and guanylyl cyclase pathways to induce chemorepulsion. Possibly as a result of not using the PI3 kinase/Akt/PKB pathway and guanylyl cyclases, AprA does not induce actin polymerization or increase the pseudopod formation rate, but rather appears to inhibit pseudopod formation at the side of cells closest to the source of AprA.

Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils.

The movement of neutrophils between blood and tissues appears to be regulated by chemoattractants and chemorepellents. Compared with neutrophil chemoattractants, relatively little is known about neutrophil chemorepellents. Slit proteins are endogenously cleaved into a variety of N- and C-terminal fragments, and these fragments are neuronal chemorepellents and inhibit chemoattraction of many cell types, including neutrophils. In this report, we show that the ∼140-kDa N-terminal Slit2 fragment (Slit2-N) is a chemoattractant and the ∼110-kDa N-terminal Slit2 fragment (Slit2-S) is a chemorepellent for human neutrophils. The effects of both Slit2 fragments were blocked by Abs to the Slit2 receptor Roundabout homolog 1 or the Slit2 coreceptor Syndecan-4. Slit2-N did not appear to activate Ras but increased phosphatidylinositol 3,4,5-triphosphate levels. Slit2-N-induced chemoattraction was unaffected by Ras inhibitors, reversed by PI3K inhibitors, and blocked by Cdc42 and Rac inhibitors. In contrast, Slit2-S activated Ras but did not increase phosphatidylinositol 3,4,5-triphosphate levels. Slit2-S-induced chemorepulsion was blocked by Ras and Rac inhibitors, not affected by PI3K inhibitors, and reversed by Cdc42 inhibitors. Slit2-N, but not Slit2-S, increased neutrophil adhesion, myosin L chain 2 phosphorylation, and polarized actin formation and single pseudopods at the leading edge of cells. Slit2-S induced multiple pseudopods. These data suggest that Slit2 isoforms use similar receptors but different intracellular signaling pathways and have different effects on the cytoskeleton and pseudopods to induce neutrophil chemoattraction or chemorepulsion.