PLCε1 suppresses tumor growth by regulating murine T cell mobilization.

Phospholipase C epsilon 1 (PLCε1) is a unique member of the phospholipase family, in that it also functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rap1. It is this function as a Rap1 GEF that gives PLCε1 an essential role in chemokine-mediated T cell adhesion. We have utilized a syngeneic tumor model, MC38 cells in C57BL/6 mice, and observed that tumors grow larger and more quickly in the absence of PLCε1. Single-cell analysis revealed an increased CD4+ /CD8+ ratio in the spleens, lymph nodes and tumors of PLCε1 knock-out tumor-bearing mice. T cells isolated from PLCε1 knock-out mice were less activated by multiple phenotypical parameters than those from wild-type mice. We additionally noted a decrease in expression of the chemokine receptors C-X-C chemokine receptor type 4 (CXCR4) and C-C motif chemokine receptor 4 (CCR4) on CD4+ T cells from the spleens, lymph nodes and tumors of PLCε1 knock-out mice compared to wild-type mice, and diminished migration of PLCε1-depleted CD3+ T cells towards stromal cell-derived factor (SDF)-1α. Based on these results, we conclude that PLCε1 is a potential regulator of tumor-infiltrating lymphocytes, functioning, at least in part, at the level of T cell trafficking and recruitment.

Galactosylceramidase deficiency causes sperm abnormalities in the mouse model of globoid cell leukodystrophy.

The classical recessive mouse mutant, "the twitcher," is one of the several animal models of the human globoid cell leukodystrophy (Krabbe disease) caused by a deficiency in the gene encoding the lysosomal enzyme galactosylceramidase (GALC). The failure to hydrolyze galactosylceramide (gal-cer) and galactosylsphingosine (psychosine) leads to degeneration of oligodendrocytes and severe demyelination. Substrate for GALC is also the galactosyl-alkyl-acyl-glycerol (GalAAG), precursor of the seminolipid, the most abundant glycolipid in spermatozoa of mammals. In this paper, we report the pathobiology of the testis and sperm in the twitcher mouse and demonstrate the importance of GALC for normal sperm maturation and function. The GALC deficit results in accumulation of GalAAG in the testis of the twitcher mouse. Morphological studies revealed that affected spermatozoa have abnormally swollen acrosomes and angulation of the flagellum mainly at midpiece-principal piece junction. Multiple folding of the principal piece was also observed. Electron microscopy analysis showed that in the twitcher sperm, acrosomal membrane is redundant, detached from the nucleus and folded over. Disorganization and abnormal arrangements of the axoneme components were also detected. These results provide in vivo evidence that GALC plays a critical role in spermiogenesis.

Activation of cell cycle regulatory proteins in the apoptosis of terminally differentiated oligodendrocytes.

There is increasing evidence that proteins normally involved in the cell cycle play a role in the regulation of neuronal apoptotic death following various insults. However, it is not clear if the same mechanisms regulate cell death of oligodendrocytes as well. In this study, we investigated the mechanism of ceramide-induced apoptosis in primary rat oligodendrocytes. We show that ceramide treatment initiates a cascade of biochemical events involving cell cycle regulatory proteins. Although at the time of induction of cell death the oligodendrocytes are postmitotic, activation of c-myc and translocation of Cdc25A into the nucleus can be demonstrated. Of particular interest are the findings of the up-regulation of PCNA and down-regulation of p21WAF1/CIP1 protein, an inhibitor of cell-cycle progression. The current results show that activation of regulatory cell-cycle proteins at the oligodendrocytes G1-S checkpoint may constitute a crucial step of the death pathway of oligodendrocytes.

Retrovirus-mediated gene transfer and galactocerebrosidase uptake into twitcher glial cells results in appropriate localization and phenotype correction.

Galactocerebrosidase (GALC) is deficient in all tissues from human patients and animal models with globoid cell leukodystrophy (GLD) or Krabbe disease. The deficiency results in decreased lysosomal catabolism of certain galactolipids including galactosylceramide and psychosine that are synthesized maximally during myelination. According to current theories, the accumulation of psychosine in humans and animals with GLD induces oligodendrocyte degeneration and myelination ceases. Transduction of oligodendrocytes from twitcher mice with a retroviral vector containing the GALC cDNA can correct the enzyme deficiency in these cells. Our data show that twitcher astrocytes and oligodendrocytes can internalize exogenous GALC, as well as donate the enzyme to the mutant glial cells. Antibodies against human GALC localized the GALC antigen in retrovirally transduced cells and cells receiving enzyme via cell to cell secretion and uptake to the lysosomal fraction. In fact immunocytochemical studies in transduced oligodendrocytes revealed that the GALC colocalizes in vesicles lysosomal-associated membrane protein-2 (LAMP2) (+). Moreover, labeling cells with anti-GALC and a marker for oligodendrocytes demonstrated that, upon differentiation, transduced, twitcher oligodendrocytes attained the normal branched process configuration, while untransduced cells show only abnormal morphology. Phenotype correction in mutant oligodendrocytes has also been observed after enzyme transfer. These studies indicate that GALC activity supplied to cultured oligodendrocytes from twitcher mice by different methods can correct the pathological phenotype of these cells.

Constitutive expression of interleukin-1beta (IL-1beta) in rat oligodendrocytes.

The RT-PCR analysis of RNA from progenitor and differentiated primary rat oligodendrocytes, and from the oligodendrocyte CG-4 cell line, shows the presence of the IL-1beta mRNA, the type I IL-1beta receptor and the IL-1 receptor accessory protein in these cells. In situ hybridization of a rat IL-1beta probe to primary progenitor and differentiated rat oligodendrocytes results in a positive signal. The double hybridization of the IL-1beta probe, together with an oligodendrocyte-specific differentiation marker, to sections of postnatal rat brain at different stages of differentiation is also positive. The double immuno-labelling technique utilized indicates coincidence of the signals on the brain slices. The results show that IL-1beta mRNA is constitutively expressed in rat brain oligodendrocytes from 1 day after birth onward. In agreement with this observation, CG-4 cells, primary progenitor and differentiated rat oligodendrocytes are positively stained by antibodies against IL-1beta. Postnatal brain slices from 1 and 4 day old and adult rats, labelled with a double immunofluorescence technique, are also stained by antibodies against IL-1beta. This signal coincides with that of antibodies against oligodendrocyte-specific surface markers. We conclude that IL-1beta is constitutively expressed in rat brain progenitor and differentiated oligodendrocytes.

Transduction of cultured oligodendrocytes from normal and twitcher mice by a retroviral vector containing human galactocerebrosidase (GALC) cDNA.

Krabbe disease or globoid cell leukodystropy is a lysosomal disorder caused by a deficiency of galactocerebrosidase (GALC) activity. This results in defects in myelin that lead to severe symptoms and early death in most human patients and animals with this disease. With the cloning of the GALC gene and the availability of the mouse model, called twitcher, it was important to evaluate the effects of providing GALC via a retroviral vector to oligodendrocytes in culture. After differentiation, the untransduced cells from normal mice extended highly branched processes while those from the twitcher mice did not. Oligodendrocytes in culture can be readily transduced to produce much higher than normal levels of GALC activity. Transduced normal and twitcher cells formed clusters when plated at high density. Transduction of twitcher oligodendrocytes plated at lower density, followed by differentiation, resulted in some cells having a completely normal appearance with highly branched processes. Other cells showed retraction and fragmentation. Perhaps over expression of GALC activity may be detrimental to oligodendrocytes. These studies demonstrate that the phenotype of twitcher oligodendrocytes can be corrected by providing GALC via gene transfer, and this could lead the way to future studies to treat this disease.

Localization of human follicle-stimulating hormone in the testis.

Localization of the follicle-stimulating hormone (FSH) molecule and its receptor (FSHR), as well as the role of FSH in Sertoli cell mitosis and maturation, has been demonstrated by several investigators in human and murine testis by detecting the localization of anti-FSH antibodies or [(131)I]-labeled FSH and by detecting FSH receptor (FSHR) mRNA by in situ hybridization, or FSHR by anti-FSHR antibodies. The presence of FSH in germinal cells is controversial or, in humans, excluded. We have investigated the distribution of the human FSH molecule and its receptor in human and mouse testicular cells under different experimental conditions, at the submicroscopical level, by using a better antigenicity conservative procedure. Thus, the distribution of FSH and of the messenger RNA for its receptor in Sertoli cells has now been clarified. In germinal cells, our observations demonstrate the presence of FSH and the FSHR mRNA: the first on the plasma membrane and in endocytotic vesicles, and the second scattered in the cytoplasm. The cells presenting the higher amount of positivity ranged from spermatogonia to spermatocytes, including round spermatids. Penetration was by the endocytosis via membrane vesicles in which the FSHR is present, whereas its messenger is largely present in the cytoplasm and is responsible for the binding and subsequent internalization of the FSH molecule. As a control, human FSH was administered in vitro to the Y1 mouse cell line, which was stably transfected with cDNA for FSHR and devoid of endogenous FSH. The FSH molecule has been localized by monoclonal antibodies on plasma membranes and vesicles, and the FSHR mRNA was found scattered in the cytoplasm after in situ hybridization. We can now conclude that FSH is present in Sertoli cells and in round germinal cells, both expressing the FSHR. FSH penetrates in a similar way in both kinds of cells via endocytosis, and is therefore subsequently localized in the same membranous organelles.

New insights into the interaction between the gp120 and the HIV receptor in human sperm (human.sperm/gp120/galactoglycerolipid/antigalactosylceramide/seminolip id/spermatogonia).

The human immunodeficiency virus (HIV) can infect some cell types which lack CD4. Galactosylceramide, a glycolipid present in the nervous system and colonic epithelial cells, has been implicated in the virus entry in these cells. Our data demonstrate that the HIV surface glycoprotein gp120 binds to the galactosyl-alkyl-acylglycerol (GalAAG), a glycolipid structurally related to galactosylceramide present on the surface membrane of the spermatozoa. In this paper, we review our previous data and further confirm the specificity of the interaction between this galactoglycerolipid and the gp120. Consistent with the structural similarity to galactosylceramide, the sperm GalAAG is capable of specifically binding the gp120. The specificity of the binding of antibodies antigalactosylceramide and the gp120 to the sperm extract and to the purified GalAAG fraction prepared from the same extract has been demonstrated utilizing an ELISA assay which favors sensitivity and specificity. Immunofluorescence and immunoelectron microscopy data show a different localization for the GalAAG and its sulfated form the seminolipid (SGalAAG). The GalAAG is preferentially localized in the equatorial segment and the middle piece of the sperm tail, while the seminolipid is widely distributed on the membrane of the spermatozoa. These data indicate that human sperm express on their surface membrane a glycolipid similar in structure to galactosylceramide, the receptor for HIV identified in the CD4 cells, that could function as a HIV receptor and possibly be implicated in its transmission.

Induction of intracellular ceramide by interleukin-1 beta in oligodendrocytes.

The sphingomyelin pathway has been implicated in mediating the effect of several extracellular agents leading to important biochemical and cellular changes. The aim of this investigation is to study interleukin-1 beta (IL-1 beta) signaling in oligodendrocytes. For this purpose, the CG4 oligodendrocyte cells were differentiated and incubated with IL-1 beta. This treatment induced a time- and dose-dependent increase of the endocellular ceramide. To mimic the effect of the elevation of endogenous ceramide, the CG4 cells were treated with the ceramide analogue C2-ceramide. Cell survival, measured with the MTT assay, showed that, by increasing the concentration of ceramide, up to 40% of CG4 cells were dying within 6 h, similar data were obtained with the primary differentiated oligodendrocytes. Condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies indicated that apoptosis was the cause of death. Surprisingly, long-term exposure (72 h) to increasing concentrations of IL-1 beta, which increases intracellular ceramide, did not induce oligodendroglial cell death. These results show that an increase of intracellular ceramide is not sufficient to induce apoptosis in oligodendrocytes and that IL-1 beta signaling through the ceramide pathway in these cells can mediate functions other than programmed cell death.

Human sperm and spermatogonia express a galactoglycerolipid which interacts with gp120.

Sexual transmission is a major mode of spread of HIV-1 although the mechanisms involved remain to be elucidated. The role of spermatozoa as carriers of the HIV is supported by recent publications, while the expression of the CD4 on the membrane of the sperm has not yet been demonstrated. The data reported in this paper show that a glycolipid molecule, most likely the galactosyl-alkyl-acylglycerol, structurally similar to galactosylceramides, is present on the surface membrane of the spermatozoa. Consistent with a structure similar to galactosylceramide, the sperm glycolipid is capable of binding the gp120 as demonstrated utilizing an improved ELISA assay which favors sensitivity and specificity. Immunocytochemistry of testicular tissue shows the presence of this glycolipid on the membrane of immature germ cells, preferentially in the spermatogonia. These data indicate that human sperm express a glycolipid similar in structure to the receptor for HIV described on the CD4- neural and colonic epithelial cell lines, and moreover suggest that this glycolipid could also function as HIV receptor and possibly be implied in its transmission. The demonstration that this molecule is also expressed by the spermatogonia suggests its involvement in the interaction of the HIV with spermatogonia, as recently reported, and could explain the inhibition of spermatogenesis observed in AIDS patients.