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Title: Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography

Abstract

Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Here, using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Authors:
 [1];  [2];  [3];  [3];  [3];  [1];  [1];  [4];  [3]; ORCiD logo [1]
  1. Baylor College of Medicine, Houston, TX (United States)
  2. Univ. of Texas Health Science Center at Houston, Houston, TX (United States)
  3. Univ. of California, San Francisco, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  4. Univ. of Texas Health Science Center at Houston, Houston, TX (United States); Univ. of Texas at Houston Graduate School of Biomedical Sciences, Houston, TX (United States); Central South Univ., Hunan (People's Republic of China)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1413703
Grant/Contract Number:
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Journal of Cell Science
Additional Journal Information:
Journal Volume: 129; Journal Issue: 18; Journal ID: ISSN 0021-9533
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Cryogenic soft X-ray tomography; Red blood cell; Sickle cell disease; Sphingosine kinase inhibitor; Red cell morphology

Citation Formats

Darrow, Michele C., Zhang, Yujin, Cinquin, Bertrand P., Smith, Elizabeth A., Boudreau, Rosanne, Rochat, Ryan H., Schmid, Michael F., Xia, Yang, Larabell, Carolyn A., and Chiu, Wah. Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography. United States: N. p., 2016. Web. doi:10.1242/jcs.189225.
Darrow, Michele C., Zhang, Yujin, Cinquin, Bertrand P., Smith, Elizabeth A., Boudreau, Rosanne, Rochat, Ryan H., Schmid, Michael F., Xia, Yang, Larabell, Carolyn A., & Chiu, Wah. Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography. United States. doi:10.1242/jcs.189225.
Darrow, Michele C., Zhang, Yujin, Cinquin, Bertrand P., Smith, Elizabeth A., Boudreau, Rosanne, Rochat, Ryan H., Schmid, Michael F., Xia, Yang, Larabell, Carolyn A., and Chiu, Wah. 2016. "Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography". United States. doi:10.1242/jcs.189225. https://www.osti.gov/servlets/purl/1413703.
@article{osti_1413703,
title = {Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography},
author = {Darrow, Michele C. and Zhang, Yujin and Cinquin, Bertrand P. and Smith, Elizabeth A. and Boudreau, Rosanne and Rochat, Ryan H. and Schmid, Michael F. and Xia, Yang and Larabell, Carolyn A. and Chiu, Wah},
abstractNote = {Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Here, using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.},
doi = {10.1242/jcs.189225},
journal = {Journal of Cell Science},
number = 18,
volume = 129,
place = {United States},
year = 2016,
month = 8
}

Journal Article:
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  • An examination was made of the O/sub 2/ affinity, Bohr effect, transmembrane pH gradient, mean cell hemoglobin concentration, and red blood cell sickling at half O/sub 2/ saturation in whole sickle cell (SS) and normal (AA) blood during CO/sub 2/ titration and acid-base titration at three Pco/sub 2/ levels, 10, 40, and 80 mm Hg. The CO/sub 2/-induced Bohr effect of SS blood was considerably larger than normal (maximum, 0.91, referred to cell pH) and similar to that found with acid-base titration at Pco/sub 2/ of 40. In contrast to AA blood, SS blood showed an increased O/sub 2/ affinitymore » when Pco/sub 2/ was raised from 40 to 80, and at half O/sub 2/ saturation showed biphasic or sigmoid Bohr curves, a fall in transmembrane pH gradient with rising Pco/sub 2/, and an absence of the normal cell volume increase at low pH and Pco/sub 2/. Sickling of SS cells at half O/sub 2/ saturation was partly inhibited by increasing Pc/sub 2/, particularly in the higher pH ranges. These complex differences in the behavior of SS blood are interpreted in terms of the balancing of several effects: the lowering of hemoglobin O/sub 2/-affinity by polymerization, low pH and increased CO/sub 2/ binding, inhibition of hemoglobin S polymerization by CO/sub 2/ binding to ..beta../sup s/-chain amino termini, differences between hemoglobin S and A in competitive binding of CO/sub 2/ and 2,3-diphosphoglycerate at different pH levels, and an increased net negative charge exhibited by intracellular deoxyhemoglobin S polymers. From a clinical standpoint, in the absence of hypoxia or acidosis, an increased blood Pco/sub 2/ might have a beneficial effect by inhibiting red blood cell sickling, whereas a metabolic acidosis, with low blood pH and Pco/sub 2/, would be very hazardous.« less
  • Natural killer (NK) cells play an important role in the innate immune response. Interleukin-18 (IL-18) is a well-known interferon-gamma (IFN-{gamma} inducing factor, which stimulates immune response in NK and T cells. Sphingosine kinase (SPHK) catalyzes the formation of sphingosine 1-phosphate (S1P), which acts as a second messenger to function as an anti-apoptotic factor and proliferation stimulator of immune cells. In this study, to elucidate whether SPHK is involved in IL-18-induced IFN-{gamma} production, we measured IL-18-induced IFN-{gamma} production after pre-treatment with SPHK inhibitor (SKI) in NK-92MI cells. We found that IL-18-induced IFN-{gamma} expression was blocked by SKI pre-treatment in both mRNAmore » and protein levels. In addition, the increased IFN-{gamma} production by stimulation with IL-18 is mediated through both SPHK and p38 MAPK. To determine the upstream signals of SKI and p38 MAPK in IL-18-induced IFN-{gamma} production, phosphorylation levels of p38 MAPK was measured after SKI pre-treatment. As a result, inhibition of SPHK by SKI blocked phosphorylation of p38 MAPK, showing that SPHK activation by IL-18 is an upstream signal of p38 MAPK activation. Inhibition of SPHK by SKI also inhibited IL-18-induced IFN-{gamma} production in human primary NK cells. In conclusion, SPHK activation is an essential factor for IL-18-induced IFN-{gamma} production via p38 MAPK.« less
  • Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.