Prosaposin ablation inactivates the MAPK and Akt signaling pathways and interferes with the development of the prostate gland

Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, Quebec, Canada (H3A 2B2)

Asian J Androl 2003 Mar; 5: 57-63             

Keywords: mouse; prostate; prosaposin; mitogen activating phosphokinases (MAPKs)

Abstract

The recent development of a prosaposin -/- mouse model has allowed the investigation of the role of prosaposin in the development of the male reproductive organs. A morphometric analysis of the male reproductive system of 37 days old mice revealed that prosaposin ablation produced a 30 % reduction in size and weight of the testes, 37 % of the epididymis, 75 % of the seminal vesicles and 60 % of the prostate glands. Light microscopy (LM) showed that smaller testis size from homozygous mutant mice was associated with reduced spermiogenesis. Both, dorsal and ventral lobules of the prostate glands were underdeveloped in the homozygous mutant. LM analysis also showed that prostatic alveoli were considerably smaller and lined by shorter epithelial cells in the homozygous mutant. Smaller tubular diameter and shorter undifferentiated epithelial cells were also observed in seminal vesicles and epididymis. In the efferent ducts of the homozygous mutant mice, the epithelium was composed exclusively of ciliated cells in contrast to the heterozygotes, which showed the presence of nonciliated cells. Radioimmunoassays demonstrated that testosterone levels were normal or higher in mice with the inactivated prosaposin gene. Immunostaining of prostate sections with an anti-androgen receptor antibody showed that the epithelial cells lining the alveoli express androgen receptor in both the heterozygous and homozygous tissue. Similarly, sections immunostained with antibodies to the phosphorylated MAPKs and Akts strongly reacted with tall prostatic secretory cells in prostate from heterozygous mouse. On the other hand, the epithelial cells in the homozygous prostate remained unstained or weakly stained. These findings demonstrate that inactivation of the prosaposin gene affected the development of the prostate gland and some components of the MAPK pathway.

1 Introduction

Prosaposin is a non-enzymatic factor that promotes the intracellular degradation of sphingolipids by specific hydrolases [1]. It is also found extracellularly in the nervous and male reproductive systems where it is linked to the activation of intracellular signaling pathways [2-4]. In the lysosomes, prosaposin is processed into four 10-15 kDa saposins (A-D). Saposins activate the hydrolysis of sphingolipids (SLs) with short oligo-saccharide chains by specific acidic hydrolases [1]. The deficiency of saposin B is linked to a variant form of metachromatic leukodystrophy [5] and the deficiency of saposin C is linked to a variant form of Gaucher's disease [1]. Gaucher's disease is considered the most prevalent

lysosomal storage disorder. Deficiencies of saposin A and D have not yet been reported. However, a genetic disease caused by the complete absence of prosaposin is related to a multiple glycosphingolipid elevation including lactosyl ceramidosis [6, 7]. Thus, the lysosomal form of prosaposin plays a role in the hydrolysis of SLs [1, 5].

SLs are components of plasma and Golgi membranes of eukaryotic cells. SLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain, that faces the cisternal lumen of the Golgi apparatus [8-11]. In the plasma membrane the catabolism of sphingolipids is involved in the generation of well known lipophilic intermediates that are involved in the transmission of extracellular signals to intracellular regulatory systems [11].

In addition to its well-documented role in the hydrolysis of SLs, secreted prosaposin has been reported to have neurotrophic activity [2-4]. Prosaposin is abundant in cerebrospinal fluid and is capable of stimulating neurite outgrowth in murine cells and choline acetyl-transferase (ChAT) activity in human neuroblastoma cells [2-4, 12]. A 12 amino acid sequence in the N-terminal end of saposin C has been implicated in the activation of the mitogen activated protein kinase (MAPK) pathway [4, 12].

The objective of this review is to examine the role of prosaposin and sphingolipids in the activation of the MAPK pathway and in the development of the prostate. The role of other regulators of prostate growth will be discussed as well. Finally, this report will also examine the effect of prosaposin ablation in the mouse prostate.

2 Prosaposin and the activation of the MAPK pathway

Prosaposin appears not only to exhibit neurotrophic activity [2] but also to be an essential factor for cell growth, differentiation and survival of the prostatic epithelium and the epithelial lining of other reproductive organs [13]. Prosaposin deficient mice exhibit a severe reduction of prostate size due to the presence of under-veloped alveoli and the absence of secretory cells [7, 13].

The trophic region of prosaposin encompasses 12 amino acids within the functional C domain [4, 12, 14]. Preliminary evidence suggests, that to exert its trophic effect, prosaposin must bind to a high affinity receptor which activates the mitogen-activated kinase pathway (MAPK) [2]. MAPK is a general name for a family of serine/threonine kinases that play an important role in cell signaling. They are activated by a variety of ligands and receptors including tyrosine kinases and G-protein-coupled receptors [2]. The extracellular signal regulated protein kinases ERK1 (p44 MAPK) and ERK 2 (p42 MAPK) are part of the MAPK family. Activation of ERKs can be the result of linear signaling cascades from growth factor receptors, adaptor proteins, guanine nucleotide exchange factors, Ras, Raf 1, and MAPK-activating kinases such as MEK [2]. Emerging data indicate that signaling proteins such as phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) can also phosphorylate MEK and ERKs independent of the Ras pathway. Therefore, the activation of the MAPKs is often complex and involves multiple signaling systems. Recent evidence in Schwann and PC12 cells suggests that prosaposin induces the entry of these cells into the S-phase of the cell cycle and prevents apoptosis by activating ERKs and sphingosine kinase via a pertussis toxin-sensitive G protein associated receptor [2, 15]. Pro-saposin also prevents programmed cell death of rat cerebellar granule neurons in culture by activating PI3K and Akt [2]. In the NS20Y and SHSY5Y cell lines prosaposin also operates through a G protein associated receptor, possibly the GO-a [15, 16].

The serine / threonine kinases Ras and PAK have emerged as the molecules that can converge signaling on the Raf-ERK pathway. Receptor-mediated activation of Raf, the upstream MAPK kinase in the ERK cascade, is Ras dependent and involves recruitment of the kinase to the plasma membrane. On the other hand PAK which is a Cdc 42 and Rac target, can phosphorylate MEK 1 on Ser298 in a region that mediates the interaction of MEK 1 with Raf. Activation of PAK has been shown to be dependent upon PI3K-dependent activation of Akt. This evidence emphasizes the notion that activation of MAPKs depends on multiple signaling pathways and also on signal convergence [17].

Activated ERK can enter the nucleus and phosphorylate transcription factors providing the link between cell surface receptor-mediated events and nuclear induction of gene expression. In the nucleus activated ERK promotes the transcription and the activity of transcription factors c-fos, c-myc, c-jun and p21 [18, 19]. The c-myc proto-oncogene encodes a short-lived transcription factor that promotes cell proliferation and suppresses growth arrest by the regulation of appropriate growth target genes [20]. The c-myc transcription factor is essential for cell proliferation [21] and overexpression of c-myc is associated with the inability of cells to withdraw from the cell cycle, resulting in uncontrolled cellular proliferation. In addition, c-myc can induce delayed late genes, such as cyclins D1, E and A [22, 23]. Hence proto-oncogene proteins normally act at critical steps in normal growth factor mitogenic signaling pathways. Overexpression or constitutive activity of mitogenic proto-oncogenes singly or in combination may cooperate and lead to transformation of cells and thus play a causal role in oncogenesis. In summary, the potential role of prosaposin in the activation of the MAPK pathway has been presented in a simplified manner (Figure 1). Finally, multiple evidence from various laboratories suggest that different signaling MAPK pathways have biological versatility in promoting complex biological responses such as cell proliferation and cell transformation.

Figure 1.  Simplified model of prosaposin activation of the MAPK signaling pathway.  The model is based on data from several laboratories.  The prosaposin receptor is still unknown.

Glycolipids are constituents of biological membranes which are composed of a carbohydrate moiety linked to a hydrophobic aglycon. They are divided into glyco-glycerolipids abundant in bacteria and plants, and glycosphingolipids (GSL), abundant in eukaryotic cells [8]. GSLs contain a hydrophobic ceramide moiety that acts as membrane anchor and a hydrophilic, extracellular oligosaccharide chain. Ceramide consists of a long chain amino alcohol, D-erythro-sphingosine, which is acylated with a fatty acid. Ceramide is also a structural component of sphingomyelin, a sphingolipid not linked to carbohydrates. GSLs are heterogeneous with respect to both their carbohydrate and ceramide portions. Variations in the type, number, and linkage of sugar residues within the oligosaccharide chain, give rise to the wide range of naturally occurring sphingolipids. Sialic acid-containing GSLs (gangliosides), are abundant on neuronal cells. GSL patterns change with cell growth, differentiation, viral transformation, ontogenesis and oncogenesis [8, 9]. Part of the plasma membrane destined for degradation is endocytosed and trafficked through the endosomal compartment to reach lysosomes [8, 9]. The composition of sphingolipids entering lysosomes depends on the cell type. Neuronal plasma membranes are rich in gangliosides while oligodendrocytes and Schwann cells have a higher content of galactosylceramide and sulfatide [8, 9]. Within lysosomes, hydrolyzing enzymes sequentially cleave off the sugar residues. Ceramide first and then sphingosine are finally produced. Sphingosine leaves lysosomes to re-enter the biosynthetic pathway [6, 8, 11]. For GSLs with long carbohydrate chains of more than four sugar residues, the presence of an enzymatically active lysosomal hydrolase is sufficient for degradation in vivo. However, degradation of membrane bound GSLs with short oligosaccharide chains requires the cooperation of lysosomal hydrolases and saposins.

Catabolism of plasma membrane sphingolipids generates lipophilic intermediates which are involved in the transmission of extracellular signals to intracellular regulatory systems [10, 11]. For example, hydrolysis of sphingomyelin to ceramide can be induced by certain ligands and receptors in various cell types. The identity of cellular targets of ceramide and other molecules downstream the signal flow is not known but several target proteins in the MAPK pathway are currently under investigation[10, 11]. In addition, a growing body of evidence suggests a mechanism of Ras-MAPK activation by sphingosine-1P activated protein kinases [10].

Because of the dual role of prosaposin (i.e., lysosomal activator of hydrolases and trophic factor), it is unclear which of these two mechanisms is more important in the development of the prostate. The inactivation of the prosaposin gene not only causes the loss of the trophic activity of prosaposin, but also the suppression of sphingolipid hydrolysis which may alter the distribution of lipophilic second messengers in the plasma membrane.

4 Regulators of prostate growth

The prostate gland develops from a complex interplay between mesenchymal and epithelial tissues [24]. The prostate evolves late in gestation as a consequence of signaling events between the urogenital sinus epithelium and the urogenital sinus mesenchyme (UGM). In rodents, the prostatic buds are formed during embryo-genesis, while the full ductal branching occurs postnatally. Androgens are important for all aspects of prostate development. During embryogenesis, androgen-dependent signaling events in the UGM give rise to paracrine factors that act on local epithelial cells to induce morphogenesis and ductal branching [24]. During puberty

and adulthood, androgen-dependent signaling events in the epithelium are required for full differentiation and production of secretory proteins [24]. In adult males, the prostate is subject to two primary age-related diseases, prostate cancer and benign prostatic hyperplasia. Prostatic cancer is currently the most commonly diagnosed neoplasm in men [25]. The disease follows a progression from an early, organ-defined disease, which may be clinically asymptomatic and treatable by prostatectomy or androgen ablation therapy, to a highly invasive, androgen-independent, metastatic disease for which there is no current effective therapy or cure [26]. Most genes that are involved in normal and abnormal prostate growth and development are important in cell proliferation and/or differentiation. Proto-oncogenes such as c-met or bcl-2 are believed to be important in the metastatic properties of prostate cancer and in the development of androgen independence [26, 27]. Transcription factors may also exert their effects in a prostate-specific manner such as the androgen receptor (AR). Amplification of AR is not involved in tumorigenesis but occur in advanced-stage prostate cancer [28].

The role of various growth factors on prostate growth has been examined. Growth factors appear to be local substances responsible for mediating the mesenchymal and epithelial interactions important for prostate development. Although no single growth factor family has been demonstrated to have a direct role in prostate cancer, it is clear that growth factor pathways are altered during prostatic carcinogenesis. Some evidence points to the role of growth factors: (1) isolated prostatic epithelial or stromal cells respond to growth factors in vitro [29]; (2) Overexpression of growth factors in transgenic mice perturbs prostatic growth and development [30, 31]; (3) Prostatic cells express growth factor receptors [32, 33, 34]; (4) Androgens directly influence the expression of growth factors [35, 36]. Following androgen withdrawal, the production of stimulatory growth factors, EGF, IGF and FGF by prostate cells decreases whereas the expression of TGFb-1 and TGFb-2 receptors increases [18, 37-39]. The net effect of these growth factors alteration is prostatic involution. Androgen replacement restores normal EGF, IGF and FGF levels and the prostate recovers its original size [40, 41].

5 Effects of prosaposin inactivation in the male reproductive system

The development of a prosaposin -/- mouse model allowed the investigation of the role of prosaposin in different tissues [6]. Inactivation of the prosaposin gene caused accumulation of lactosylceramide, glucosylcera-mide, digalactosylceramide, sulfatide, ceramide and globotriaosylceramide in lysosomes of affected cells [6]. Ultrastructural analysis of these cells showed accumulation of undigested membranes in multivesicular bodies. Mice generally die at day 35~40 after birth due to neurological defects [6].

A morphometric analysis of the male reproductive system of 37 days old mice revealed that disruption of the prosaposin gene produced a 30 % reduction in size and weight of the testes, 37 % of the epididymis, 75 % of the seminal vesicles and 60 % of the prostate glands (Figure 2). Light microscopy (LM) showed that smaller testes from homozygous mutant mice were associated with reduced spermiogenesis, and that late spermatids were the most affected cells. Underdeveloped prostate glands in homozygous mutant were characteristic of both, dorsal and ventral lobules (Figure 2). In addition, LM analysis showed that the tubuloalveolar glands were considerably smaller and that the prostatic epithelial cells were shorter in the homozygous mutant (Figure 3). Smaller tubular diameter and shorter undifferentiated epithelial cells were also observed in seminal vesicles and epididymis. In the efferent ducts of the homozygous mutant mice, the epithelium was composed exclusively of ciliated cells in contrast to the heterozygotes, which showed a majority of nonciliated cells [7, 13].

Figure 2. Dorsal prostate (solid stars) and seminal vesicle (open stars). Note that the inactivation of the prosaposin gene (left picture) caused a significant reduction in size of the prostate and seminal vesicles. The right picture illustrates a control normal prostate and the seminal vesicle of a heterozygous (-/+) mouse. Both pictures are flanked by their respective drawings to permit a better appreciation of the developmental effect of prosaposin ablation.

Figure 3. Immunoperoxidase staining of prostate sections with an anti-phospho-Akt antibobody. Note that the prostatic epithelial cells of prosaposin KO are unstained (A). On the other hand, the supranuclear region of secretory epithelial cells of heterozygous (control) prostate (B) is heavily stained. 400

Radioimmunoassay of blood samples from homo-zygous mutant, heterozygous and control mice (n=6) revealed that testosterone levels were normal or higher in mice with the inactivated prosaposin gene. Moreover, the immunostaining of prostate sections with an anti-androgen receptor antibody indicates that the epithelial cells lining the alveoli express androgen receptor in both the heterozygous and homozygous tissue. Involution of the prostate gland and other male reproductive organs appears to be independent of androgen levels and androgen receptor. Thus, inactivation of the prosaposin gene seems to interfere with the proliferative activities of prostatic epithelial cells [7, 13]. Due to the dual role of prosaposin as a lysosomal activator of hydrolases and a trophic factor, it is unclear what is the determining cause for the lack of development of the prostate. Inactivation of the gene not only causes the loss of prosaposin trophic activity but also affects the lysosomal degradation of sphingolipids which may alter the distribution of lipophilic second messengers such as ceramide in the plasma membrane. In fact, catabolism of plasma membrane sphingolipids generates lipophilic intermediates, which are involved in the transmission of extracellular signals to intracellular regulatory systems [42]. For example, hydrolysis of sphingomyelin to ceramide can be induced by certain ligands and receptors in various cell types. In general, ceramide appears to mediate cell differentiation, and apoptosis [11]. The identity of the cellular targets of ceramide and other molecules downstream the signal flow is not fully known [11] but a strong body of evidence indicates that ceramide is also involved in the activation of the MAPK pathway [43]. To examine if this was the case, prostate sections from homozygous and hetero-zygous mice were immunostained with two monoclonal antibodies, which recognize the phosphorylated and non-phosphorylated forms of MAPKs. Sections immuno-stained with the antibody to the non-phosphorylated form of MAPKs reacted with all epithelial cells in heterozygous and homozygous prostates. Sections immunostained with the antibody to the phosphorylated MAPKs strongly reacted with tall prostatic secretory cells in prostate from heterozygous mouse. On the other hand, the epithelial cells in the homozygous prostate remained unstained. We have recently found that the Akt signaling pathway is also affected by the disruption of the prosaposin gene. Prostate sections immunostained with an antibody to a phospho-threonine 308 form of Akt reacted with tall prostatic secretory cells in prostate from heterozygous mouse. Conversely, the epithelial cells of the homozygous prostate were weakly stained with the Akt antibody (Figure 3). The Akt signaling pathway, activated by several growth factors, promotes cell survival by inhibiting apoptosis through phosphorylation and deactivation of proapoptotic proteins [43]. Therefore, the involution of the prostate and other reproductive organs may be explained, in part, by the activation of apoptotic proteins due to the inhibition of Akt [43]. Since apoptosis is not readily observed in the epithelium lining the alveoli of the prostate of 38-40 day old mutant mice, it is possible that this process occurs gradually during prenatal and/or posnatal development. Nevertheless, this observation requires further verification. In summary, our results demon-strated for the first time, that inactivation of the prosa-posin gene affects the development of the prostate gland and some components of the MAPK and Akt signaling pathways [7].

The molecular basis of prostate cancer is still poorly understood but it is clear that the limitations of androgen-deprivation therapy will be circumvented by the development of more specific growth factors, signal transduction and angiogenesis inhibitors. Thus, the study of this unique protein may provide information on the role of prosaposin in normal and pathological conditions.

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Correspondence to: Carlos R. Morales, Department of Anatomy and Cell Biology, McGill University, 3640 University Street, Montreal, Quebec, Canada H3A 2B2
Tel: +1-514-398 6398,  Fax: +1-514-398 5047
E-mail: carlos.morales@mcgill.ca
Received 2003-02-28   Accepted 2003-03-07