Regulation of the amount of ceramide-1-phosphate synthesized in differentiated human podocytes

Abstract

Sphingolipids have important functions as structural components of cells but they also function as signaling molecules regulating different cellular processes such as apoptosis, cell proliferation, cell migration, cell division and inflammation. Hence, a tight regulation of the sphingolipid homeostasis is essential to maintain proper cellular functions. Mammalian ORMDL proteins are orthologues of the yeast ORM1/2 proteins, which regulate ceramide synthesis in yeast. ORMDL proteins inhibit serine palmitoyltransferase (SPT), the enzyme regulating a rate-limiting step of the sphingolipid pathway to control the levels of ceramides and other sphingolipids. Sphingomyelinase phosphodiesterase like 3b (SMPDL3b) is a glycosylphosphatidylinositol (GPI) anchored protein in the plasma membrane (PM) and determines membrane fluidity in macrophages. We previously showed that differential expression of SMPDL3b alters the availability of Ceramide-1-phosphate (C1P) in human podocytes, which are terminally differentiated cells of the kidney filtration barrier. This observation lead us to investigate if SMPDL3b controls C1P availability in human podocytes by interfering with ceramide kinase (CERK) expression and function. We found that SMPDL3b interacts with CERK and can bind to C1P in vitro. Furthermore, CERK expression is reduced when SMPDL3b expression is silenced. These observations led us to propose that one of the mechanisms by which SMPDL3b influences the amount of C1P available in the podocytes is by interfering with the function of CERK thereby maintaining a balance in the levels of the C1P in podocytes.

Keywords: Ceramide-1-phosphate; Podocytes; SMPDL3b; Sphingolipids.

Fig 1.. SMPDL3b interacts with CERK.

(A) Immunoprecipitation using HEK293 cells transfected with FLAG-hCERK, HA-hSMPDL3b, HA-hSMPDL3bH135A and empty FLAG vector (negative control) showing the interaction of SMPDL3b with CERK. N=3. (B) Endogenous immunoprecipitation demonstrating the interaction of endogenous SMPDL3b with CERK using pooled glomeruli from C57Bl/6 mice. Lane 1, Dyna beads coupled with SMPDL3b antibodies. Lane 2, Dyna beads coupled with IgG antibodies (negative control). N=2. Band corresponding to SMPDL3b is shown with red arrow.

Fig 2.. SMPDL3b regulates C1P levels in podocytes.

Liquid chromatography mass spectrometry analysis (LC-MS) of A) Total ceramide-1-phosphate (C1P), B) Total ceramides content in Ctrl, siSMPDL3b and SMPDL3b OE differentiated human podocytes. ****P<0.0001 ***P=0.0003, **P<0.01. ANOVA, followed by Tukey's multiple comparisons test. All data are presented as mean ± SE. N=3.

Fig 3.. SMPDL3b binds to ceramide-1-phosphate (C1P).

PVDF membranes spotted with C1P and Sph were incubated with Anti-FLAG M2 affinity gel purified proteins from HEK293 cells transfected with A) FLAG-SMPDL3b vector or B) FLAG-empty vector (negative control) or C) Without any protein/vector, sphingosine (Sph). N=3.

Fig 4.. SMPDL3b affects CERK expression.

(A) Heat map of Illumina RNA sequence analysis of mRNA from SMPDL3b overexpressing (SMPDL3b OE) compared to control (Ctrl) human podocytes. Genes related to the sphingolipid pathway that are upregulated are shown in blue and genes that are downregulated are shown in orange. Details of Gene expression are shown in supplementary Table 3. Genes that have significant FDR value (<0.05) were shown. (B) Bar graph analysis of mRNA expression of genes connected to SL pathway, analyzed by qRT-PCR in differentiated human podocytes. (C) Western blot and bar graph analysis of the fold change in protein expression of SMPDL3b (shown with red arrow), CERK, SPTLC3, SMPD1 and ACER2 to GAPDH. ****p<0.0001, ***p<0.0006, **p=0.0062, *p<0.05; ANOVA, followed by Tukey's multiple comparisons test. All data are represented as mean ± SE. N=3.

Fig 5.. Ceramide-1-Phosphate exhibits a feedback inhibition on the expression of CERK.

A) Western blot and B) Bar graph analysis of the fold change in CERK expression in Ctrl, siSMPDL3b and SMPDL3b OE podocytes with and without 20μM C16-C1P treatment for 24 hrs. Cells were serum-starved overnight followed by treatment with C16-C1P for 24 hours. **P<0.009,*P<0.05; two tailed t-test. All data are represented as mean ± SE. N=4.

Fig 6.. Sphingolipid metabolism and regulation of C1P levels in human podocytes.

Human podocytes control the increase in C1P levels by a mechanism in which C1P exerts a negative feedback inhibition on the expression of ceramide kinase (shown with red arrow). SMPDL3b when overexpressed, blocks CERK access to ceramide and leads to decrease in the levels of C1P (shown with brown arrow). De novo sphingolipid pathway is shown with black arrows. The salvage pathway is shown with blue arrow. All enzymes are shown in green color. SPT, serine palmitoyl transferase; SMS, sphingomyelin synthetase; SMase, sphingomyelinase; CerS, ceramide synthase; SK, spingosine kinase; S1PP, spingosine-1-phosphate phosphatase; S1P lyase, spingosine-1-phosphate lyase; SMPDL3b, sphingomyelin phosphodiesterase acid-like 3b; CERK, ceramide kinase; GCS, glycosylceramide synthase; GCase, glycosylceramidase; GalCS, galactosylceramide synthase; GalCDase, galactosylceramidase.