Taqman analysis of enzymes involved in Cer generation and degradation

PEST domain

DMPE- DMPE-TMR

E- LDL and Ox-LDL loading

5.4. Taqman analysis of enzymes involved in Cer generation and degradation

To verify the gene array data and to get informations about the regulation of further enzymes involved in Cer generation and degradation, Taqman realtime PCR was performed. ApoE3/3 and apoE4/4 macrophages during E-LDL and Ox-LDL loading and HDL₃deloading were used and enzymes involved in sphingolipid metabolism were analyzed. In the following results fold changes are related to the non differentiated state of the monocytes.

Serine palmitoyltransferase 1 and 2, which were not present on the chip were decreased during differentiation in both genotypes (Fig.54A,B). During lipid loading both serine palmitoyltransferases were stronger upregulated in apoE4/4 than in apoE3/3 macrophages.

In apoE3/3 macrophages serine palmitoyltransferase 1 was 1.7 fold increased during E-LDL and 2.1 fold during Ox-LDL loading (Fig.54A) while in apoE4/4 macrophages serine palmitoyltransferase 1 was 4.7 fold increased during E-LDL and 3.8 fold increased during Ox-LDL loading (Fig.54B). Serine palmitoyltransferase 2 was 1.3 fold upregulated during E-LDL and 1.5 fold upregulated during Ox-E-LDL loading in apoE3/3 macrophages (Fig.54C) while in apoE4/4 macrophages serine palmitoyltransferase 2 was 2.4 fold upregulated during E-LDL and 4.3 fold during Ox-LDL loading (Fig.54D). Sphingomyelin synthase 1 and 2 (SMS1 and 2) were decreased during differentiation in both genotypes (Fig.54E,F). During lipid loading SMS1 and 2 were stronger upregulated in apoE4/4 than in apoE3/3 macrophages. ApoE3/3 macrophages showed a 1.5 fold upregulation of SMS1 during E-LDL and a 1.3 fold upregulation during Ox-LDL loading (Fig.54E) and SMS2 was 1.6 fold upregulated during E-LDL and Ox-LDL loading (Fig.54G). Whereas in apoE4/4 macrophages SMS1 was 4.6 fold upregulated during E-LDL and 1.6 fold during Ox-LDL loading (Fig.54F) and SMS2 increased up to 5.9 fold during E-LDL loading and 3.8 fold during Ox-LDL loading (Fig.54H). Acid Sphingomyelinase (acidSMase) showed only a significant regulation in apoE3/3 macrophages (Fig.54I). It was induced during differentiation up to 2.8 fold. E-LDL loading led to a -1.7 fold decrease while Ox-LDL loading led to a 3.1 fold increase (Fig.54I).

In apoE4/4 macrophages during differentiation a 1.4 fold increase of acid SMase could be observed with no further changes during lipid loading (Fig.54J). The increase during Ox-LDL loading in apoE3/3 macrophages of acid SMase which degrade SM to Cer could lead to an increased Cer formation in apoE3/3 but not in apoE4/4 macrophages. Neutral SMase was regulated similarly in both genotypes (Fig.54K,L). It was decreased during differentiation and increased during lipid loading. During E-LDL loading the upregulation was lower than during LDL loading with 2.6 fold in the apoE3 and 3.9 fold in the apoE4 genotype while with Ox-LDL loading it was 5.2 fold in the apoE3 and 4.7 fold in the apoE4 genotype (Fig.54K,L).

Neutral SMase activation associated factor (NSMAF) was decreased during differentiation in both genotypes (Fig.54M,N). During lipid loading it was stronger induced in apoE4/4 compared with apoE3/3 macrophages. E-LDL loading of apoE3/3 macrophages showed a

1.3 fold upregulation of NSMAF while in apoE4/4 macrophages were 7.2 fold upregulated (Fig.54M,N). Ox-LDL loading led to a 2.3 fold upregulation of NSMAF in apoE3/3 and to a 11 fold upregulation in apoE4/4 macrophages (Fig.54M,N). Acid ceramidase was increased during differentiation in apoE3/3 macrophages while it was slightly decreased in apoE4/4 macrophages (Fig.54O,P). During lipid loading acid ceramidase was 1 fold decreased in the apoE3 genotype compared to MCSF while it was 11.8 fold increased in the apoE4 genotype during E-LDL loading and 7.9 fold during Ox-LDL loading (Fig.54O,P). The increase of acid ceramidase during lipid loading in the apoE4 genotype could enhance ceramide degradation leading to a lower level of Cer with decreased generation of GSLs. Glucosylceramidase was not regulated during differentiation in both genotypes (Fig.54Q,R). During E-LDL loading it was 1.8 fold upregulated in apoE3 and 1.3 fold in apoE4 macrophages. During Ox-LDL loading it was 3.6 fold upregulated in apoE3/3 macrophages while in apoE4/4 macrophages it was only 1.6 fold upregulated (Fig.54Q,R). This could lead to a stronger increase of GlcCer during Ox-LDL loading in apoE4/4 than in apoE3/3 macrophages. This could allow the conclusion that there may be a defect in the traffic of Cer and GSLs from the Golgi to the cell surface as a reason for the lower Cer and GSL surface expression in the apoE4 genotype.

UDP-glucose ceramide glucosyltransferase was not regulated during differentiation but during E-LDL and Ox-LDL loading the upregulation was higher in apoE4/4 than in apoE3/3 macrophages (Fig.54S,T). E-LDL loading led in apoE3/3 macrophages to a 2.1 fold upregulation while in apoE4/4 macrophages a 13 fold upregulation could be observed. Ox-LDL loading of apoE3/3 macrophages led to a 3.2 fold upregulation while Ox-Ox-LDL loading of apoE4/4 macrophages led to 15.2 upregulation of UDP-glucose ceramide glucosyltransferase (Fig.54S,T). Upregulation of UDP-glucose ceramide glucosyltransferase which transfers Cer to GlcCer could lead to an increased degradation of Cer with an increase of GlcCer in apoE4/4 macrophages. These data could also lead to the conclusion that there may be a defect in the traffic of Cer and GSLs from the Golgi to the cell surface.

With HDL₃ deloading of E-LDL loaded cells of the apoE3/3 genotype almost no change could be observed of serine palmitoyltransferase 1 and 2 mRNA expression (Fig.54A,C).

While in the apoE4/4 genotype the mRNA expression of serine palmitoyltransferase 1 and 2 was decreased from 4.7 and 4.3 fold respectively up to 1.3 fold (Fig.54B,D). Deloading of Ox-LDL loaded cells led in the case of apoE3/3 macrophages to a decrease from 2.1 to 1 fold of serine palmitoyltransferase 1 while no change of serine palmitoyltransferase 2 mRNA expression could be observed (Fig.54A,C). In the case of apoE4/4 macrophages during Ox-LDL deloading an increase from 4.7 fold to 5.4 fold of serine palmitoyltransferase 1 (Fig.54B) and an increase from 2.4 fold to 7 fold of serine palmioyltransferase 2 (Fig.54D) could be detected. SMS1 was upregulated in apoE3/3 macrophages during E-LDL deloading from 1.1 fold to a 2.1 fold while there was no change of SMS2 mRNA expression (Fig.54E,G). In

apoE4/4 macrophages during E-LDL deloading a decrease of mRNA expression of SMS1 from 4.6 fold to 1.2 fold and in the case of SMS2 from 5.9 fold to 1.7 fold could be observed (Fig.54F,H). During Ox-LDL deloading SMS1 was upregulated from 1.1 fold to 1.7 fold and SMS2 was decreased from 1.6 fold to 1.2 fold in apoE3/3 macrophages (Fig.54E,G). While in the apoE4 genotype an upregulation from 1.6 fold to 3.5 fold of SMS1 and a decrease from 3.8 fold to 3 fold of SMS2 could be observed during Ox-LDL deloading (Fig.54F,H). The mRNA expression of acid SMase was decreased in the apoE3 genotype from 1.7 fold to 1 fold during E-LDL deloading (Fig.54I) while in the apoE4 genotype acid SMase was slightly increased during E-LDL deloading from 1.5 fold to 1.8 fold (Fig.54J). During Ox-LDL deloading a decrease of acid SMase from 3.1 fold to 0.4 fold could be observed in apoE3/3 (Fig.54I) and from 1.5 fold to 0.9 fold in apoE4/4 macrophages (Fig.54J). With HDL3

deloading the mRNA expression of neutral SMase was decreased in both genotypes. In the apoE3 genotype during E-LDL deloading from 2.6 fold to 0.9 fold (Fig.54K) and in the apoE4 genotype from 3.9 to 1 fold (Fig.54L). During Ox-LDL deloading a decrease from 5.2 fold to 0.3 fold took place in the apoE3 (Fig.54K) and from 4.7 fold to 1.3 fold in the apoE4 genotype (Fig.54L). NSMAF decreased during Ox-LDL deloading with HDL3 from a higher level in apoE4/4 macrophages (Fig.54M,N). During E-LDL deloading almost no change could be observed in the apoE3 genotype while NSMAF was downregulated in the apoE4 genotype (Fig.54M,N). Acid ceramidase was decreased during HDL₃ deloading from a higher level in apoE4/4 than in apoE3/3 macrophages (Fig.54O,P). The upregulation of glucosylceramidase in apoE3/3 macrophages was reversed with HDL₃ deloading (Fig.

54Q,R). During E-LDL deloading from 1.8 fold to 1.1 fold and during Ox-LDL deloading from 3.6 to 0.4 fold (Fig.54Q) while in apoE4/4 macrophages almost no change of glucosylceramidase mRNA expression could be detected during E-LDL and Ox-LDL deloading (Fig.54R). UDP-glucose ceramide glucosyltransferase was decreased during HDL3 deloading. In apoE3/3 macrophages during E-LDL deloading from 3.2 fold to 0.8 fold and during Ox-LDL deloading from 2.1 fold to 0.7 fold (Fig.54S). In apoE4 macrophages UDP-glucose ceramide glucosyltransferase was decreased from a higher level during HDL₃ deloading. During E-LDL deloading it was decreased from 15.2 fold and during Ox-LDL deloading from 13 fold up to 1.1 fold (Fig.54T).

relative mRNA expression B. serine palmitoyltransferase 1 D. serine palmitoyltransferase 2

relative mRNA expression F. sphingomyelin synthase 1

E-LDL

relative mRNA expression B. serine palmitoyltransferase 1 D. serine palmitoyltransferase 2

relative mRNA expression F. sphingomyelin synthase 1

E-LDL

relative mRNA expression G. sphingomyelin synthase 2

E-LDL

relative mRNA expression H. sphingomyelin synthase 2

E-LDL

relative mRNA expression G. sphingomyelin synthase 2

E-LDL

relative mRNA expression H. sphingomyelin synthase 2

E-LDL