Renal Mesangial Cells Isolated from Sphingosine Kinase 2 Transgenic Mice Show Reduced Proliferation and are More Sensitive to Stress-Induced Apoptosis

Abstract

Background/Aims: Sphingosine 1-phosphate (S1P) is considered as a key molecule regulating various cell functions including cell growth and death. It is produced by two sphingosine kinases (SK) denoted as SK-1 and SK-2. Whereas SK-1 has been extensively studied and has been appointed a role in promoting cell growth, the function of SK-2 is controversial, and both pro-proliferative and pro-apoptotic functions have been suggested. In this study we investigated whether renal mesangial cells isolated from transgenic mice overexpressing the human Sphk2 gene (hSK2-tg) showed an altered cell response towards growth-inducing and apoptotic stimuli. Methods: hSK2-tg mice were generated by using a Quick Knockin^R strategy. Renal mesangial cells were isolated by a differential sieving method and further cultivated in vitro. Lipids were quantified by mass spectrometry. Protein expression was determined by Western blot analysis, cell proliferation was determined by ³H-thymidine incorporation, and apoptosis was determined by a DNA fragmentation ELISA. Results: We show here that kidneys and mesangial cells from hSK2-tg mice express the hSK2 as well as the endogenous mouse mSK2. hSK2 and mSK2 predominantly resided in the cytosol of quiescent transgenic cells. However, S1P accumulated strongly in the nucleus and only minimally in the cytosol of transgenic cells. Functionally, hSK2-tg cells proliferated less than control cells under normal growth conditions and were also more sensitive towards stress-induced apoptosis. On the molecular level, this was reflected by reduced ERK and Akt/PKB activation, and upon staurosporine treatment, by a sensitized mitochondrial pathway as manifested by reduced anti-apoptotic Bcl-XL expression and increased cleavage of caspase-9, downstream caspase-3 and PARP-1. Conclusion: Altogether, these data demonstrate that SK-2 exerts an antiproliferative and apoptosis-sensitizing effect in renal mesangial cells which suggests that selective inhibitors of SK-2 may promote proliferation and reduce apoptosis and this may have impact on the outcome of proliferation-associated diseases such as mesangioproliferative glomerulonephritis.

A Rheostat of Ceramide and Sphingosine-1-Phosphate as a Determinant of Oxidative Stress-Mediated Kidney Injury

“…Table 1 summarizes the localization of enzymes that regulate ceramide formation and SphKs in the kidney [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 ]. Regarding subcellular localization of enzymes, sphingomyelin synthase (SMS)1/2 is predominantly in the Golgi and to a small extent in the plasma membrane [ 20 ].…”

“…SphK1 is predominantly localized in the cytosol [ 49 ], microsomes, plasma membranes and nuclei of kidney [ 49 ], the lysosomes of podocytes [ 52 ], and the cytosol, plasma membrane, mitochondria, lysosomes/peroxisomes, Golgi and ER of RTCs [ 55 ]. SphK2 is predominantly localized in the cytosol of kidney, mesangial cells (MCs) and RTCs [ 50 , 53 , 55 ] and nuclei of MCs [ 53 ].…”

“…Overexpression of SphK2 promoted apoptosis in β cells by interacting with Bcl-xL, contributing to the progression of DN [ 234 ]. Transfection of SphK2 also rendered MCs and RTCs more sensitive to TNF-α-induced mitochondrial dysfunction which produces ROS and ceramide, by decreasing Bcl-xL [ 53 ]. In addition, the cooperation of SphK2 with C8-BID stimulated the expression of Bax and MOMP, leading to apoptosis.…”

“…In contrast, MCs overexpressing SphK2 were susceptible to apoptosis caused by LPS and TNF-α, which produce ROS and ceramide by inhibiting ERK/Akt/protein kinase B (PKB) and Bcl-xL [ 53 ]. In addition, S1P/S1PR1 activated Akt and eNOS which counteracted ROS production, ameliorating hypoxia-induced EC apoptosis [ 225 ].…”

“…In contrast, the overexpression of SphK2 reduced MC proliferation by inhibiting ERK/Akt/PKB [ 53 ]. SphK2 knockout increased the expression of SphK1/S1PR3 and ERK/PI3K/Akt due to an increase in SphK1 activity, leading to increased proliferation/migration, whereas inhibitors of ERK and of S1PR3 abolished SphK1-induced MC proliferation [ 256 ].…”