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Abstract

Sphingolipids serve as structural elements of cells and as lipid second messengers. They regulate cellular homeostasis, mitogenesis, and apoptosis. Sphingolipid signaling may also be important in various pathophysiologies such as vascular injury, inflammation, and cancer. Serine palmitoyltransferase (SPT) catalyzes the condensation of serine with palmitoyl-CoA, the first, rate-limiting step in de novo sphingolipid biosynthesis. This integral microsomal membrane protein consists of at least two subunits, SPT1 and SPT2. In this study we analyzed the expression of SPT1 and SPT2 in normal human tissues. Strong SPT1 and SPT2 expression was observed in pyramidal neurons in the brain, in colon epithelium, and in mucosal macrophages. However, SPT2 expression was more prominent than SPT1 in the colon mucosal macrophages, the adrenomedullary chromaffin cells and endothelium, and in the uterine endothelium. SPT2 was localized in both nuclei and cytoplasm of the adrenomedullary chromaffin cells, whereas SPT1 was primarily cytoplasmic. These observations link enhanced SPT expression to proliferating cells, such as the lung, stomach, intestinal epithelium, and renal proximal tubular epithelium, and to potentially activated cells such as neurons, chromaffin cells, and mucosal macrophages. A baseline expression of SPT, established by this study, may serve as a measure for aberrant expression in various disease states.

Keywords

serine palmitoyltransferase human tissues immunohistochemistry antibodies

Membrane lipid compositions are highly characteristic of different membranes and can depend on the physiological state of the cell, thus making it important to understand the regulation of these phenomena (Merrill 1983). Sphingolipids are ubiquitous components of eukaryotic but not prokaryotic cell membranes. In addition to providing structural integrity to cell and organelle membranes, there is emerging evidence for the involvement of sphingolipids in regulating various cellular functions. Sphingolipid metabolic intermediates, such as sphingosine, sphingosine-1-phosphate, ceramide, sphingoylphosphorylcholine, psychosine, and lysogangliosides, are either important second messengers or cell regulatory molecules. Ceramide, synthesized de novo, is believed to promote apoptosis (Xu et al. 1998; Perry et al. 2000). This underlines the potential importance of sphingolipid metabolism in physiologically important phenomena such as senescence (Venable et al. 1995), growth and differentiation (Okazaki et al. 1990), tumor suppression, tissue development, injury, and atrophy (reviewed in Hannun 1997). Sphingosine has multiple targets within cells. For example, it can inhibit protein kinase C (Hannun and Bell 1989; Lavie et al. 1990; Yamada et al. 1993; Natarajan et al. 1994), activate sphingosine-dependent kinases (Megidish et al. 2000), and regulate cell proliferation (Fatatis and Miller 1999). Another sphingolipid mediator, sphingosine-1-phosphate (S-1-P), interacts with members of the endothelial differentiation gene, G-protein-coupled receptor (Edg receptor) family, to induce cell proliferation and survival (Zhang et al. 1991; Lee et al. 1998; An et al. 2000; Hla et al. 2000). S-1-P is also important in platelet activation (Yatomi et al. 1997) and the regulation of adherens junction formation (Lee et al. 1999), and therefore affects cell trafficking and vascular permeability (Carmeliet et al. 1999; Dejana et al. 1999). S-1-P is also important for extracellular matrix assembly, neurite retraction (Postma et al. 1996), cell motility, tumor invasion (Sadahira et al. 1992), and programmed cell death. Furthermore, S-1-P affects Ca2⁺ mobilization (Ghosh et al. 1994; Mattie et al. 1994) and K⁺ influx (Bunemann et al. 1995; van Kop-pen et al. 1996) within cells.

The backbone of various sphingolipids is generated from the long-chain bases sphinganine, sphingosine and, in yeast, phytosphinganine. The first unique and committed reaction to long-chain base synthesis involves the condensation of L-serine with a fatty acid acyl-CoA to generate 3-ketodihydrosphingosine by the enzyme serine palmitoyltransferase [SPT; palmitoyl-CoA; L-serine C-palmitoyltransferase (decarboxylating)] (Merrill 1983). An integral microsomal membrane protein (Mandon et al. 1992), SPT is composed of at least two subunits, SPT1 and SPT2. The catalytic subunit of SPT is believed to be SPT2, whereas the regulatory activity is believed to be the SPT1 subunit. In yeast, both LCB1 and LCB2 subunits are required for LCB activity (Nagiec et al. 1994) and a third component, Tsc3p, is essential for optimal LCB function (Gable et al. 2000).

Recently, SPT expression has been closely linked to pathophysiological conditions. Procedures such as angioplasty result in vascular injury and, in response to injury, a cascade of events collectively known as restenosis is initiated. An increase in SPT1 and SPT2 expression has been reported in proliferating vascular smooth muscle cells and fibroblasts in balloon-injured rat carotid arteries (Uhlinger et al. 2001). Upregulation of SPT subunits has also been observed in proliferating fibroblasts, transformed cell lines, and in various human tumors (unpublished observations). The association of increased SPT expression with injury makes it a potential therapeutic target.

Because SPT activity is altered by a change in the physiological state of the cell, it is imperative to determine the basal levels of this enzyme in normal tissues. The distribution of the SPT1 and SPT2 subunits may serve as a potential marker of cell activity in which high levels of the enzyme may reflect increased activity (e.g., neuronal transmission, exocytosis) or cell proliferation. The SPT1 and SPT2 levels determined in normal tissues and cell types might then be used to analyze cell types in abnormal states such as cancers, inflammation, and vascular injury. Therefore, in this investigation we examined the distribution of SPT1 and SPT2 in normal human tissues using immunohistochemistry (IHC).

Materials and Methods

Antibody Production

Rabbit polyclonal antibodies to the SPT subunits were generated using antigenic peptide sequences predicted by the algorithm of Hopp/Woods. The peptides utilized for antibody production against the human SPT1 subunit (GenBank protein accession number CAA69941) were KLQERSDLT-VKEKEEC, corresponding to residues 45–59, and KEQE-IEDQKNPRKARC, corresponding to residues 222-236. The peptides used as antigens for the human SPT2 subunit (GenBank protein accession number CAA69942) were CGK-YSRHRLVPLLDRPF, corresponding to residues 538-552, and CGDRPFDETTYEETED, corresponding to residues 549-561. A cysteine and glycine were added to the amino terminus of these peptides to allow KLH conjugation and decreased steric hindrance for the coupling. Rabbit polyclonal antibodies were raised against both peptides separately for each SPT subunit. The resulting immune sera were pooled and the mixed polyclonal antisera used as the source of antibody against the specific SPT subunit.

Western Analysis

The specificity of the rabbit anti-SPT1 or rabbit anti-SPT2 polyclonal antibody was evaluated by immunoblotting analysis. Microsomal membranes from HEK 293 cells stably transfected with both SPT1 and SPT2 were prepared as described by Williams et al. (1984). Fifteen micrograms of microsomal membrane protein was fractionated in each of the six lanes of an SDS-polyacrylamide gel. After transfer to a nitrocellulose membrane, the membrane was probed with 2 μg/ml dilution of SPT1- or SPT2-specific polyclonal serum described above. The antigen specificity of the polyclonal serum was determined by preincubating with the serum with 20 μg/ml antigenic peptides overnight at 4C before probing the immunoblot. Bound antibody was detected with an alkaline phosphatase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnologies; Santa Cruz, CA).

Immunohistochemistry

Commercial human normal and tumor checkerboard tissue slides (DAKO, Carpinteria, CA; Biomeda, Foster City, CA) were deparaffinized, hydrated, and processed for routine IHC as previously described (D'Andrea et al. 1998). Briefly, slides were microwaved in Target (DAKO), cooled, placed in PBS, pH 7.4, then placed in 3.0% H₂O₂. Slides were processed through an avidin-biotin blocking system according to the manufacturer's instructions (Vector Labs; Burlin-game, CA) and then placed in PBS. All reagent incubations and washes were performed at room temperature. Normal blocking serum (Vector Labs) was placed on all slides for 10 min. After brief rinsing in PBS, primary antibodies (Table 1) were placed on slides for 30 min. The slides were washed and biotinylated secondary antibodies, goat anti-rabbit (goat anti-rabbit) or horse anti-mouse (monoclonal antibodies), were placed on the tissue sections for 30 min (Vector Labs). After rinsing in PBS, the avidin-horseradish peroxidase-biotin complex reagent (ABC; Vector Labs) was added for 30 min. Slides were washed and treated with the chromogen 3,3'-diaminobenzidine (DAB; Biomeda), rinsed in dH₂O, and counterstained with hematoxylin. Monoclonal antibodies to vimentin (Table 1) were used to demonstrate tissue antigenicity and reagent quality. Negative controls included replacement of the primary antibody with the same species IgG isotype nonimmune serum (Table 1). The specificity of the primary SPT antibodies was again determined by preincubating the polyclonal sera overnight with the anti-genic peptides as described above.

Double Immunohistochemical Labeling

Protocols for simultaneous double immunohistochemical labeling (IHC:IHC) have been previously published (D'Andrea et al. 1999) and are similar to those cited for single IHC labeling except that the slides were not processed for counter-staining after the second chromogen step of the first antigen detection protocol. Instead, the slides were placed in PBS and the second antigen was detected by an alkaline phosphatase-Fast Red system. The primary antibody was placed on the slides for 30 min at RT. After brief washing, the secondary biotinylated antibody was added for 30 min at RT. The slides were first washed in PBS and then the streptavidin-alkaline phosphatase reagent was placed on the slides for 30 min at RT. After washing, the Fast Red chromogen (DAKO) was placed on the slides twice for 5 min. Then the slides were processed for routine counterstaining in hematoxylin, washed, and coverslipped in a water-based mounting medium (DAKO) for viewing under a BX-50 Olympus light microscope.

Multiple controls were performed to ensure correct interpretation of the labeling on the slides. The primary antibodies were substituted with the proper species isotype to control for the detection systems. On another set of controls, the first primary was omitted and the second primary antibody was processed, and vice versa.

Results

Specificity of SPT1 and SPT2 Antibodies

Rabbit polyclonal antibodies specific for the two human SPT subunits were generated as described in Materials and Methods. The specificity of the antibodies is demonstrated in the immunoblot shown in Figure 1a. Microsomal membrane fractions obtained from SPT stably transfected HEK cells were resolved by SDS-PAGE and the Western blot was probed with either preimmune serum or the SPT-specific polyclonal antibodies in the presence or absence of competing peptides. Single immunoreactive bands of the expected molecular weights (Weiss and Stoffel 1997) were observed, specifically M_r ∼55 kD for SPT1 (Figure 1a, Lane 3) and M_r ∼65 kD for SPT2 (Figure 1a, Lane 6). Preincubating the polyclonal antibodies with the antigenic peptides before probing the immunoblot competitively inhibited detection of SPT1 (Figure 1a, Lane 4) and SPT2 (Figure 1a, Lane 7). No nonspecific binding was observed with the preimmune serum (Figure 1a, Lanes 2 and 5).

Table 1

Primary antibodies

Name	Type	Titer	Vendor
Nonimmune serum	Polyclonal, IgG	2.0 μg/ml	Vector Labs
Nonimmune serum	Monoclonal, IgM	2.5 μg/ml	Vector Labs
Preimmune serum	Polyclonal, IgG	2.0 μg/ml	JJPRD
SPT1	Polyclonal, IgG	2.0 μg/ml	J&JPRD
SPT2	Polyclonal, IgG	2.0 μg/ml	J&JPRD
Smooth muscle actin	Monoclonal, IgM	2.0 μg/ml	DAKO
Vimentin	Monoclonal, IgM	2.0 μg/ml	DAKO

The specificity of polyclonal antibodies in IHC was also determined (Figures 1b-1e). Preincubation of the polyclonal antisera with a ten fold excess of antigenic peptides before probing the slides competitively inhibited detection of SPT1 and SPT2 (Figures 1b and 1d).

Tissue Distribution of SPT1 and SPT2

SPT1 and SPT2 protein expression in normal human tissues was analyzed using IHC. Formalin-fixed, paraffin-embedded tissues were used in a multi-tissue format to eliminate potential staining artifacts such as slide-to-slide and run-to-run variability. Table 1 lists the positive and negative controls in addition to the experimental antibodies. Positive labeling was defined by the strength of brown staining and was scored according to the following criteria: no immunoreactivity was scored as negative (N); light-brown immunoreactivity was scored as weak (W); brown immunoreactivity was scored as moderate (M); and dark-brown immunoreactivity was scored as strong (S). The negative controls did not produce observable labeling.

The distribution of SPT1 and SPT2 in human tissues is presented in Table 2. In general, the vascular endothelium and smooth muscle cells were moderately immunopositive for SPT1 and SPT2. Except for the ovarian epithelium, the epithelial cells in all tested tissues were moderate to strongly immunopositive for SPT1 and SPT2. In addition, mucosal macrophages from the colon, lung, and stomach were strongly immunopositive for SPT1 and SPT2. In the spleen, the macrophages and the polymorphonuclear cells (PMNs) stained positive for both SPT1 and SPT2, but no reactivity was observed in the lymphocytes. The colon, lung, prostate, stomach, thyroid, uterus, and vascular tissues were moderate to strongly immunopositive for SPT1 and SPT2. However, SPT1 and SPT2 were either weakly present or completely undetectable in the skin and heart tissues using the above described protocol. Figures 2–6 show some of the human tissues tested for SPT1 and SPT2 expression by IHC.

Normal brains were immunolabeled with preimmune serum (Figure 2a), SPT1 (Figure 2b)-, and SPT2 (Figure 2c)-specific antibodies. In the cerebral cortex, the pyramidal neurons (arrowheads) showed positive immunoreactivity for SPT1 and SPT2. Both SPT1 and SPT2 were localized in the neuronal cytoplasm and the expression levels of both subunits appeared similar. Purkinje cells in the human cerebellum were moderately immunopositive for both SPT1 and SPT2 (data not shown). In contrast, SPT1 and SPT2 were not detectable in other neuronal cell types, such as astro-cytes, microglia, and oligodendritic cells.

Figure 1

Competition assay to show specificity of the anti-SPT1 and anti-SPT2 polyclonal antibodies using immunoblotting analysis (a) and immunohistochemistry (b-e). Fifteen micrograms of microsomal membrane proteins from HEK293 cells stably transfected with SPT1 and SPT2 was fractionated on six lanes of an SDS-polyacrylamide gel (a). After transferring the proteins to a nitrocellulose membrane, the six lanes were cut apart and probed separately with a 2 μg/ml dilution of G-protein purified antibody from the SPT1 and SPT2 pre-immune sera (Lanes 2 and 5), the SPT1 and SPT2 antisera (Lanes 3 and 6), or the SPT1 and SPT2 antisera pre-incubated with the respective competing antigenic peptides at 20 μg/ml (Ag) (Lanes 4 and 7). Bound antibody was detected using a 1:5000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG. The figure shows a band of the expected molecular weight for SPT1 (55 kD) and SPT2 (65 kD). To assess the specificity of the SPT polyclonal antibodies in immunohistochemistry, normal human colon was immunolabeled with anti-SPT1 and anti-SPT2 antibodies (b-e) in the presence (b,d) or absence (c,e) of SPT1 and SPT2 antigenic peptides. The presence of competing SPT1 and SPT2 antigenic peptides inhibited the detection of SPT1 and SPT2 in the tissue. Original magnification X400.

Table 2

Immunolocalization of SPT1 and SPT2 in normal human tissues a

Tissue	Cell types	SPT1	SPT2
Adrenal	Cortex	N	N
	Medulla (chromaffin cells)	S	S
Brain	Neurons	S	S
	Astrocytes	N	N
	Oligodendrites	N	N
	Purkinje cells	M	M
Breast	Epithelium	M	M
Colon	Epithelium	M	M
	Mucosal macrophages	S	S
	Smooth muscle	M	M
Heart	Cardiocytes	N	N
	Endomysium	W	W
Kidney	Glomerular endothelial cells	M	M
	Epithelium, distal tubule	N	N
	Epithelium, proximal tubule	S	S
Liver	Hepatocytes	N	N
	Endothelium	M	M
Lung	Epithelium	M	M
	Endothelium	M	M
	Macrophages (dust cells)	S	S
Ovary	Epithelium	N	N
	Cortical stroma	S	S
	Myofibroblasts	M	M
Pancreas	Islets of Langerhans	N	N
	Acinar cells	M	M
Prostate	Epithelium	M	M
	Smooth muscle	M	M
Skin	Epidermis	W	W
	Dermis	N	N
Spleen	Sinusoid endothelium	S	S
	Lymphocytes	N	N
	Macrophages, PMNs	M	M
Stomach	Epithelium	S	S
	Mucosal macrophages	S	S
	Smooth muscle	M	M
Testis	Seminiferous epithelium	W	W
	Sertoli cells	W	W
	Leydig cells	M	M
Thyroid	Epithelium	M	M
Uterus	Epithelium	M	M
	Myometrium	M	M
Vascular	Endothelium	M	M
	Smooth muscle	M	M

^aNumber of labeled cells for SPT1 and SPT2 in a X100 viewing field in normal human tissues (n = 2-10). Negative (N), no labeled cells; weak (W), 1-10 labeled cells; moderate (M), 11-20 labeled cells; strong (S), >20 labeled cells. This table does not reflect differences observed between SPT1 and SPT2 immunolabeling.

In the human colon (Figure 3), epithelial cells (small arrowheads) and macrophages (large arrowheads) stained positive for SPT1 (Figure 3b) and SPT2 (Figure 3c). As in the neurons, expression of both SPT1 and SPT2 was mainly cytoplasmic. Compared to the moderate expression of SPT2 in the epithelial cells, the mucosal macrophages exhibited a much stronger immunoreactivity to SPT2. No immunoreactivity was observed in any cell type on staining with the preimmune serum (Figure 3a). The high expression of SPT in mucosal macrophages in the colon (Figures 3b and 3c) and stomach and dust cells (alveolar macrophages) (Table 2) may be due to the fact that these macro-phages are associated with tissues that are prone to environmental exposure and may therefore have been activated.

Figure 4 shows normal human adrenal glands immunolabeled with either preimmune serum (Figure 4a) or SPT1 (Figure 4b)- or SPT2 (Figure 4c)-specific antibody. Chromaffin cells of the adrenal medulla (large arrowheads), vascular smooth muscle cells (arrows), and endothelium (small arrowheads) showed strong positive cytoplasmic immunoreactivity for SPT1 and SPT2. SPT1 and SPT2 expression was undetectable in the adrenal cortex. SPT2 expression in the endothe-lium and the chromaffin cells appeared higher than SPT1. In addition to the cytoplasm, SPT2 expression could be clearly observed in the chromaffin cell nuclei.

Immunolabeling of normal human kidneys with preimmune serum (Figure 5a), SPT1 (Figure 5b)- or SPT2 (Figure 5c)-specific antibody showed SPT1 and SPT2 in the proximal tubules (arrowheads). Endothelial cells of the glomerulus were also SPT1- and SPT2-immunopositive (data not presented). Interestingly, the expression of SPT1 and SPT2 was again different in the proximal tubules. SPT1 expression was diffuse in the cytoplasm, whereas SPT2 immunostaining appeared more punctate and overall weaker than SPT1. No immunoreactivity was observed with the preimmune serum. Thus far, SPT activity has been localized to the cytosolic side of the endoplasmic reticulum (Mandon et al. 1992). Note the proximity of SPT2 expression to the nucleus. Because the endoplasmic reticulum is closely associated with the nucleus, the punctate appearance of SPT2 in the renal proximal tubule epithelium suggests its association with the endoplasmic reticulum.

Several normal human uteri were similarly immunolabeled with either preimmune serum (Figure 6a) or the SPT1 (Figure 6b)- or SPT2 (Figure 6c)-specific antibodies. Uterine smooth muscle cells (large arrowheads) demonstrated similar positive immunoreactivity for SPT1 and SPT2. However, the expression of SPT2 was higher than SPT1 in the endothelial cells (small arrowheads). No immunoreactivity was observed with the preimmune serum.

Our study indicated that SPT1 and SPT2 expression was particularly strong in cells, such as the adrenal chromaffin cells, that secrete epinephrine and norepinephrine on autonomic nervous stimulation and in neurons. Because it also appeared that SPT1-and SPT2-positive labeling was observed in proliferating cell types, such as the epithelial layers in the stomach, lungs (data not shown), renal proximal tubules, and colonic lumen, double IHC labeling of the human large intestines was performed. Antibodies to PCNA (in red), a marker of cell proliferation (D'Andrea et al. 1994), were used in combination with either the SPT1 (Figure 7a)- or the SPT2 (Figure 7b)-specific antibody (in brown). Large arrowheads show the co-localization of red- and brown-labeled cells, indicating that both SPT1 and SPT2 are expressed in proliferating epithelial cells.

Discussion

Our current studies characterize the distribution of the SPT subunits SPT1 and SPT2 in normal human tissues. The differences we observed in expression of SPT1 and SPT2 indicate that the localization and expression levels of SPT may be linked to the physiological state of the cell. Proliferating cells and cells that may potentially be activated, such as mucosal macro-phages in colon or chromaffin cells in the adrenal medulla, expressed higher levels of SPT. The presence of moderate to high levels of SPT1 and SPT2 in vascular tissues suggests a role for SPT in regulating signaling pathways involving messengers like S-1-P.

The differences between SPT1 and SPT2 expression in the same cell type within the same tissue suggests that there must be specific and possibly independent functions of each subunit in enabling SPT activity. How these two subunits interact and coordinate SPT activity is still unknown. Unlike yeast, overexpression of murine SPT2 alone in human HEK293 cells results in a corresponding increase in SPT activity, whereas SPT1 alone does not increase SPT activity (Weiss and Stoffel 1997). Whether an increase in SPT2 alone is sufficient for upregulation of SPT activity remains to be seen. In addition to SPT1 and SPT2, human SPT may also have additional components like the Tsc3p protein in yeast (Gable et al. 2000). Moreover, the localization of SPT2 in the nuclei suggests that SPT2 associates with another nuclear protein(s) or is modified and transported to the nucleus. Therefore, analysis of the difference in dynamics of SPT1 and SPT2 expression will help in elucidating SPT activity.

Enzymes that regulate sphingolipid metabolism are critical in maintaining cellular homeostasis, and a disruption of their activity can lead to disease. Inhibition of ceramide synthase by fumonisin mycotoxins contaminating animal feeds results in equine leukoen-cephalomalacia and porcine pulmonary edema (reviewed in Marasas 2001). Local delivery of C₆-ceramide in rabbit carotid arteries after balloon angioplasty reduced neointimal hyperplasia by inhibiting extracellular signal-related kinase ERK and phosphorylation of protein kinase B (PKB/Akt) (Charles et al. 2000). Signaling pathways involving ERK, PKB/Akt, and the angiotensin II receptor are involved in vascular smooth muscle migration and growth. Activation of angiotensin II receptor induces de novo sphingolipid biosynthesis, leading to programmed cell death (Lehtonen Jukka et al. 1999). Lowering S-1-P production in TNF-α-induced endothelial cells by HDL reduces the expression of adhesion proteins and consequently increases protection against artherosclerosis (Xia et al. 1999). Therefore, enzymes regulating sphingolipid metabolism are key factors in controlling sphingolipid mediated regulation of cellular phenomena.

Because SPT is the key regulatory enzyme in de novo sphingolipid biosynthesis, it is expected that an alteration in SPT activity would affect sphingolipid-mediated regulation of cell function. In yeast, SPT has been implicated in heat and hyperosmolar stress responses (Buede et al. 1991; Patton et al. 1992; Jenkins et al. 1997). Cultured human keratinocytes, when UV-irradiated, upregulate SPT activity and show a corresponding increase in SPT2 mRNA and protein levels (Farrell et al. 1998). SPT activity is increased during apoptosis and governs de novo ceramide synthesis in cells treated with the chemotherapeutic agent etoposide (Perry et al. 2000). Inhibition of SPT activity by myriocin reverses the apoptotic and antiproliferative effects of a ceramide synthase inhibitor, fumonisin, in pig kidney cells LLCK-1 (Riley et al. 1999).

Figure 2

SPT expression in human brain tissue. Normal human brain was immunolabeled with preimmune serum (a), SPT1 (b), and SPT2 (c) antibodies. Pyramidal neurons (arrowheads) in the cerebral cortex show positive intracellular immunoreactivity for SPT1 (b) and SPT2 (c). Other supporting neuronal cell types, such as astrocytes, oligodentritic cells, and microglia, do not express detectable levels of SPT1 or SPT2. Original magnification X600.

Figure 3

SPT expression in human colon. Normal human large intestine was immunolabeled with preimmune serum (a), SPT1 (b), and SPT2 (c) antibodies. Epithelial cells (small arrowheads) and macrophages (large arrowheads) show positive intracellular immunoreactivity for SPT1 (b) and SPT2 (c). Original magnification X600.

Figure 4

SPT expression in human adrenal tissue. Normal human adrenal gland was immunolabeled with preimmune serum (a), SPT1 (b), and SPT2 (c) antibodies. Chromaffin cells (large arrowheads), vascular smooth muscle cells (arrows), and endothelium (small arrowheads) show positive immunoreactivity for SPT1 (b) and SPT2 (c). No detectable SPT1 or SPT2 immunolabeling is present in the supporting stromal fibroblasts. Original magnification X600.

Figure 5

SPT expression in human kidney tissue. Normal human kidney was immunolabeled with preimmune serum (a), SPT1 (b), and SPT2 (c) antibodies. Proximal tubules (arrowheads) show positive immunoreactivity for SPT1 (b) and SPT2 (c). SPT1 presents diffuse intracellular labeling patterns in the epithelial cells, which is different from the punctate labeling pattern of SPT2 in the same cell type. Original magnification X600.

Figure 6

SPT expression in human uterus. Normal human uterus was immunolabeled with preimmune serum (a), SPT1 (b), and SPT2 (c) antibodies. Stromal smooth muscle cells (large arrowheads) and endothelium (small arrowheads) show positive immunoreactivity for SPT1 (b) and SPT2 (c). Original magnification X600.

Figure 7

Co-expression of SPT1 and SPT2 with PCNA in the human colon. Double IHC labeling of human large intestine using antibodies to PCNA (red) and SPT1 (a) and SPT2 (brown) (b). Large arrowheads show the co-localization of red- and brown-labeled cells, indicating that SPT1 and SPT2 are expressed in proliferating cells. Small arrowheads show the presence of SPT1 (a) and SPT2 (b) in macrophages. Original magnification X600.

A knowledge of SPT expression in normal cells can be used to measure abnormal cellular activity in proliferative disorders such as cancers. Both the absolute level of expression of SPT and the localization of enzyme activity may be indicative of an alteration in cell physiology. The increase in SPT activity observed in pathophysiological conditions, such as vascular hyperplasia (Uhlinger et al. 2001), wound healing, and tumors (unpublished observations), suggests therapeutic potential for SPT. Inhibiting or lowering SPT activity in these conditions might affect the symptoms associated with the conditions. In porcine epithelial kidney cells, LLC-PK1, fumonisin-induced cytotoxity and antiproliferative effects were reduced on treating the cells with SPT1-specific inhibitors such as myriocin. In the same study, IP administration of myriocin to BALB/C mice reduced free sphingosine accumulation in the kidney by 60%, with no apparent clinical side effects (Riley et al. 1999). Therefore, SPT inhibitors such as myriocin may have important therapeutic potential in treatment of proliferative disorders such as cancer and may affect pathophysiologies associated with conditions such as inflammation and vascular injury.

The immunolocalization of SPT1 and SPT2 presented here is the first direct comparison of SPT1 and SPT2 expression in normal human tissues and is a critical first step towards elucidating the complexity of SPT activity in the cell. Understanding the role of these components in SPT activity is imperative in determining the regulation of the many critical sphingolipid-mediated cellular functions and responses in various disease states.

Footnotes

Acknowledgements

We express our thanks for the excellent histological and immunohistochemical expertise of Patti A. Reiser, BS, MT, HT (ASCP), Norah A. Gumula, HT (ASCP), Zabrina Thomson, MS, and Brenda M. Hertzog, BS, MT (ASCP) of the Morphometrics Department.

References

Zheng

Bleu

(2000) Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G protein-coupled receptors Edg3 and Edg5. J Biol Chem 275:288–296

Buede

Rinker-Schaffer

Pinto

Lester

Dickson

(1991) Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids [published erratum appears in J Bacteriol 1993; 175:919]. J Bacteriol 173:4325–4332

Bunemann

Brandts

zu Heringdorf

van Koppen

Jakobs

Pott

(1995) Activation of muscarinic K+ current in guinea-pig atrial myocytes by sphingosine-1-phosphate. J Physiol 489:701–777

Carmeliet

Lampugnani

Moons

Breviario

Compernolle

Bono

Balconi

. (1999) Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis. Cell 98:147–157

Charles

Sandirasegarane

Yun

Bourbon

Wilson

Rothstein

Levison

. (2000) Ceramide-coated balloon catheters limit neointima hyperplasia after stretch injury in carotid arteries. Circ Res 87:282–288

D'Andrea

Derian

Leturcq

Baker

Brunmark

Ling

Darrow

. (1998) Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J Histochem Cytochem 46:157–164

D'Andrea

Farber

Foglesong

(1994) Immunohistochemical detection of DNA topoisomerase IIa and IIb compared with detection of Ki-67, a marker of cellular proliferation, in human tumors. Applied Immunohistochem 2:177–185

D'Andrea

Rogahn

Damiano

Andrade-Gordon

(1999) A combined histochemical and double immunohistochemical labeling protocol for simultaneous evaluation of four cellular markers in restenotic arteries. Biotech Histochem 74:172–180

Dejana

Bazzoni

Lampugnani

(1999) Vascular endothelial cadherin (VE)-cadherin: only an intercellular glue? Exp Cell Res 252:13–19

10.

Farrell

Uchida

Nagiec

Harris

Dickson

Elias

Holleran

(1998) UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes. J Lipid Res 39:2031–2038

11.

Fatatis

Miller

(1999) Cell cycle control of PDGF-induced Ca2+ signaling through modulation of sphingolipid metabolism. FASEB J 13:1291–1301

12.

Gable

Slife

Bacikova

Monaghan

Dunn

(2000) Tsc3p is an 80-amino acid protein associated with serine palmitoyltransferase and required for optimal enzyme activity. J Biol Chem 275:7597–7603

13.

Ghosh

Bian

Gill

(1994) Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium. J Biol Chem 269:22628–22635

14.

Hannun

(1997) Sphingolipid second messengers: tumor suppressor lipids: eicosanoids and other bioactive lipids in cancer, inflammation and radiation. Injury 2:305–312

15.

Hannun

Bell

(1989) Functions of sphingolipids and sphingolipid breakdown products in cellular regulation [see Comments]. Science 243:500–507

16.

Hla

Lee

Ancellin

Thangada

Liu

Kluk

Chae

. (2000) Sphingosine-1-phosphate signaling via the EDG-1 family of G-protein-coupled receptors. Ann NY Acad Sci 905:16–24

17.

Jenkins

Richards

Wahl

Mao

Obeid

Hannun

(1997) Involvement of yeast sphingolipids in the heat stress response of Saccharomyces cerevisiae. J Biol Chem 272:32566–32572

18.

Lavie

Piterman

Liscovitch

(1990) Inhibition of phosphatidic acid phosphohydrolase activity by sphingosine. Dual action of sphingosine in diacylglycerol signal termination. FEBS Lett 277:7–10

19.

Lee

Thangada

Claffey

Ancellin

Liu

Kluk

Volpi

. (1999) Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 99:301–312

20.

Lee

van Brocklyn

Thangada

Liu

Hand

Menzeleev

Spiegel

. (1998) Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science 279:1552–1555

21.

Lehtonen Jukka

Horiuchi

Daviet

Akishita

Dzau

(1999) Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis. J Biol Chem 274:16901–16906

22.

Mandon

Ehses

Rother

van Echten

Sandhoff

(1992) Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver. J Biol Chem 267:11144–11148

23.

Marasas

WFO

(2001) Discovery and occurrence of the fumonisins: a historical perspective. Environ Health Perspect Suppl 109:239–243

24.

Mattie

Brooker

Spiegel

(1994) Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway. J Biol Chem 269:3181–3188

25.

Megidish

Hamaguchi

Iwabuchi

Hakomorj

(2000) Assays of sphingosine-dependent kinase for 14-3-3 protein. Methods Enzymol 312:381–387

26.

Merrill

AHJ

(1983) Characterization of serine palmitoyltransferase activity in Chinese hamster ovary cells. Biochim Biophys Acta 754:284–291

27.

Nagiec

Baltisberger

Wells

Lester

Dickson

(1994) The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis. Proc Natl Acad Sci USA 91:7899–7902

28.

Natarajan

Jayaram

Scribner

Garcia

(1994) Activation of endothelial cell phospholipase D by sphingosine and sphingosine-1-phosphate. Am J Respir Cell Mol Biol 11(2):221–229

29.

Okazaki

Bielawska

Bell

Hannun

(1990) Role of cer-amide as a lipid mediator of 1 alpha, 25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 265:15823–15831

30.

Patton

Srinivasan

Dickson

Lester

(1992) Phenotypes of sphingolipid-dependent strains of Saccharomyces cerevisiae. J Bacteriol 174:7180–7184

31.

Perry

Carton

Shah

Meredith

Uhlinger

Hannun

(2000) Serine palmitoyltransferase regulates de novo ceramide generation during etoposide-induced apoptosis. J Biol Chem 275:9078–9084

32.

Postma

Jalink

Hengeveld

Moolenaar

(1996) Sphin-gosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor. EMBO J 15:2388–2392

33.

Riley

Norred

Bacon

Meredith

Sharma

(1999) Serine palmitoyltransferase inhibition reverses anti-proliferative effects of ceramide synthase inhibiton in cultured renal cells and suppresses free sphingoid base accumulation in kidney of BALB/c mice. Environ Toxicol Pharmacol 7:109–118

34.

Sadahira

Ruan

Hakomori

Igarashi

(1992) Sphingosine 1-phosphate, a specific endogenous signaling molecule controlling cell motility and tumor cell invasiveness. Proc Natl Acad Sci USA 89:9686–9690

35.

Uhlinger

Carton

Argentieri

Damiano

D'Andrea

(2001) Increased expression of serine palmitoyltransferase (SPT) in balloon-injured rat carotid artery. Thromb Haemost 86:1320–1326

36.

van Koppen

zu Heringdorf

Laser

Zhang

Jakobs

Buenemann

Pott

(1996) Activation of a high affinity Gi protein-coupled plasma membrane receptor by sphingosine-1-phosphate. J Biol Chem 271:2082–2087

37.

Venable

Lee

Smyth

Bielawska

Obeid

(1995) Role of ceramide in cellular senescence. J Biol Chem 270:30701–30708

38.

Weiss

Stoffel

(1997) Human and murine serine-palmitoyl-CoA transferase—cloning, expression and characterization of the key enzyme in sphingolipid synthesis. Eur J Biochem 249:239–247

39.

Williams

Wang

Merrill

AHJ

(1984) Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes. Arch Biochem Biophys 228:282–291

40.

Xia

Vadas

Rye

Barter

Gamble

(1999) High density lipoproteins (HDL) interrupt the sphingosine kinase signaling pathway. A possible mechanism for protection against atherosclerosis by HDL. J Biol Chem 274:33143–33147

41.

Yeh

Chen

Sensi

Canzoniero

Choi

. (1998) Involvement of de novo ceramide biosynthesis in tumor necrosis factor-alpha/cycloheximide-induced cerebral endothelial cell death. J Biol Chem 273:16521–16526

42.

Yamada

Sakane

Imai

Takemura

(1993) Sphingosine activates cellular diacylglycerol kinase in intact Jurkat cells, a human T-cell line. Biochim Biophys Acta 1169:217–224

43.

Yatomi

Igarashi

Yang

Hisano

Asazuma

Satoh

. (1997) Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum. J Biochem 121:969–973

44.

Zhang

Desai

Olivera

Seki

Brooker

Spiegel

(1991) Sphingosine-1-phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol 114:155–167

Characterization of Serine Palmitoyltransferase in Normal Human Tissues

Abstract

Keywords

Materials and Methods

Antibody Production

Western Analysis

Immunohistochemistry

Double Immunohistochemical Labeling

Results

Specificity of SPT1 and SPT2 Antibodies

Tissue Distribution of SPT1 and SPT2

Discussion

Footnotes

Acknowledgements

References