Uteroglobin is essential in preventing immunoglobulin A nephropathy in mice.

The molecular mechanism(s) of immunoglobulin A (IgA) nephropathy, the most common primary renal glomerular disease worldwide, is unknown. Its pathologic features include hematuria, high levels of circulating IgA-fibronectin (Fn) complexes, and glomerular deposition of IgA, complement C3, Fn and collagen. We report here that two independent mouse models (gene knockout and antisense transgenic), both manifesting deficiency of an anti-inflammatory protein, uteroglobin (UG), develop almost all of the pathologic features of human IgA nephropathy. We further demonstrate that Fn-UG heteromerization, reported to prevent abnormal glomerular deposition of Fn and collagen, also abrogates both the formation of IgA-Fn complexes and their binding to glomerular cells. Moreover, UG prevents glomerular accumulation of exogenous IgA in UG-null mice. These results define an essential role for UG in preventing mouse IgA nephropathy and warrant further studies to determine if a similar mechanism(s) underlies the human disease.

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood.

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.

Induction of an enzyme in genetically deficient rats after grafting of normal liver.

Tissue from normal rat livers was grafted onto the livers of rats that were genetically deficient in bilirubin uridine diphosphate glucuronyltransferase activity. Twelve weeks after the grafting operation, the liver of the recipient rats had bilirubin uridine diphosphate glucuronyltransferase activity.

Maternal ethanol exposure induces transient impairment of umbilical circulation and fetal hypoxia in monkeys.

When ethanol was administered intravenously to pregnant monkeys, a transient but marked collapse of umbilical vasculature was observed uniformly within about 15 minutes. The ethanol-induced impairment of umbilical circulation produced severe hypoxia and acidosis in the fetus; recovery occurred during the succeeding hour. This striking interruption of feto-placental circulation may explain one of the mechanisms of mental retardation, a frequent manifestation in children afflicted with fetal alcohol syndrome.

Suppression of epididymal sperm antigenicity in the rabbit by uteroglobin and transglutaminase in vitro.

There is evidence that the mammalian female genital tract is capable of responding immunologically when challenged with alloantigens. The antigenic properties of male gametes have been well delineated. However, it is only rarely that a female mammal ever responds immunologically to the male gametic antigens as a result of coitus. When a proposed mechanism of suppression of antigenicity of epididymal spermatozoa was tested experimentally, the results indicated that two proteins (uteroglobin and transglutaminase) present in the prostate may be responsible for suppressing sperm antigenicity in the rabbit.

Response to ethanol reduced by past thiamine deficiency.

Ethanol-induced intoxication and hypothermia were studied in rats approximately 7 months after severe thiamine deficiency, when treated rats appeared to have recovered their physical health. Previously induced thiamine deficiency without prior ethanol exposure significantly decreased the area under the curve plotted for the concentration of ethanol in blood and also decreased behavioral impairment and hypothermia due to ethanol exposure. Pathophysiologic changes resulting from thiamine deficiency may contribute to both the pharmacodynamic and pharmacokinetic tolerance to ethanol in chronic alcoholics.

Severe fibronectin-deposit renal glomerular disease in mice lacking uteroglobin.

Despite myriads of biological activities ascribed to uteroglobin (UG), a steroid-inducible secreted protein, its physiological functions are unknown. Mice in which the uteroglobin gene was disrupted had severe renal disease that was associated with massive glomerular deposition of predominantly multimeric fibronectin (Fn). The molecular mechanism that normally prevents Fn deposition appears to involve high-affinity binding of UG with Fn to form Fn-UG heteromers that counteract Fn self-aggregation, which is required for abnormal tissue deposition. Thus, UG is essential for maintaining normal renal function in mice, which raises the possibility that an analogous pathogenic mechanism may underlie genetic Fn-deposit human glomerular disease.

Uteroglobin: structure, molecular biology, and new perspectives on its function as a phospholipase A2 inhibitor.

UG or blastokinin is a low molecular weight protein which is secreted by the endometrium of the rabbit during early pregnancy. Its synthesis and secretion by the endometrium are regulated by ovarian steroids, especially P. However, the protein is also produced by tracheo-bronchial, gastrointestinal, prostatic, and seminal vesicular epithelium. In the respiratory tract, UG synthesis is under glucocorticoid control. The hormonal regulation of UG synthesis in organs other than the endometrium and tracheobronchial epithelium is poorly understood. The structure of this protein and its gene has been extensively investigated while its physiological function is still unclear. Since UG, after reduction of its two disulfide bonds, has the ability to bind P and related steroids, it has been suggested that this protein is a P carrier or a P scavenger. However, the protein does not bind glucocorticoids, estrogens, or androgens and its presence in organs other than the uterus cannot be explained on the basis of its P binding. Recent data indicate that UG has other interesting biological properties. These include antichemotactic/antiphagocytic effects on macrophages, monocytes, and neutrophils, tolerogenic effect on both blastomeres and spermatozoa against recognition by maternal lymphocytes, and its ability to inhibit thrombin-induced platelet aggregation. Moreover, UG has been shown to be a potent phospholipase A2 inhibitor. The latter property could suggest a possible mechanism of some of the observed biological effects of this protein. The structural similarities of UG with phospholipase A2 and with other PLA2 inhibitory proteins like lipocortins may suggest that the physiological function of this protein may be primarily immunomodulatory through its function as a PLA2 inhibitor. The possible occurrence of similar proteins in other species, including humans, may confirm this hypothesis. Additionally, our hypothesis may lend support to the suggestion that P may be nature's immunosuppressant during pregnancy.

Tissue-specific expression of the gene coding for human Clara cell 10-kD protein, a phospholipase A2-inhibitory protein.

Clara cell 10-kD protein (cc10kD), a secretory phospholipase A2 inhibitor, is suggested to be the human counterpart of rabbit uteroglobin (UG). Because cc10kD is expressed constitutively at a very high level in the human respiratory epithelium, the 5' region of its gene may be useful in achieving organ-specific expression of recombinant DNA in gene therapy of diseases such as cystic fibrosis. However, it is important to establish the tissue-specific expression of this gene before designing gene transfer experiments. Since the UG gene in the rabbit is expressed in many other organs besides the lung and the endometrium, we investigated the organ and tissue specificity of human cc10kD gene expression using polymerase chain reaction, nucleotide sequence analysis, immunofluorescence, and Northern blotting. Our results indicate that, in addition to the lung, cc10kD is expressed in several nonrespiratory organs, with a distribution pattern very similar, if not identical, to that of UG in the rabbit. These results underscore the necessity for more detailed analyses of the 5' region of the human cc10kD gene before its usefulness in gene therapy could be fully assessed. These data also suggest that cc10kD and UG may have similar physiological function(s).

Uteroglobin gene expression in the rabbit uterus throughout gestation and in the fetal lung. Relationship between uteroglobin and eicosanoid levels in the developing fetal lung.

Uteroglobin (UG) gene encodes a cytokine-like, multifunctional, antiinflammatory protein, with potent phospholipase A2-inhibitory activity. It has been suggested that during implantation this protein protects the embryos from maternal immunological assault, facilitates the maintenance of quiescence in the uterus throughout pregnancy, prevents the onset of premature labor, and helps maintain an inflammation-free respiratory organ. This latter function of UG is suggested to be accomplished by preventing hydrolysis of surfactant phospholipids by a lung-specific phospholipase A2. Using reverse transcription polymerase chain reaction, in situ hybridization, immunofluorescence, and radioimmunoassay, we studied UG gene expression in the rabbit uterus throughout gestation and in the fetal lung. Here, we report that: (a) contrary to previous reports, UG gene expression in the rabbit uterus occurs throughout gestation with a precipitous decline just before parturition; (b) this gene expression is dramatically increased in the fetal lung with increasing gestational age; and (c) while there is an inverse relationship between the levels of UG, PGE2, and PGF2 alpha, a positive correlation was found in that of UG and leukotriene C4 in the fetal lung. Our results raise the possibility that dysregulation of UG gene expression, at least in part, may contribute to the onset of premature labor and the development of inflammatory lung disease in premature neonates.

Transketolase abnormality in cultured fibroblasts from familial chronic alcoholic men and their male offspring.

We have investigated a thiamine-dependent enzyme, transketolase, in cultured fibroblasts from 41 human subjects, including patients with alcoholism-associated Wernicke-Korsakoff syndrome (n = 3), familial chronic alcoholic males (n = 7), their sons (n = 7), nonalcoholic men (n = 7), their male offspring (n = 7), and three generations of an Amish family (n = 10) without any history of alcoholism. This study was undertaken to delineate whether transketolase abnormality (i.e., high Michaelis Menton constant (Km) for thiamine pyrophosphate), previously reported in patients with Wernicke-Korsakoff syndrome is prevalent among familial chronic alcoholic men and their sons without prior history of alcohol abuse but who are at high risk for alcoholism. Our data suggest that an inborn error (i.e., high Km of transketolase for thiamine pyrophosphate) predisposing to thiamine deficiency diseases similar to those reported in Wernicke-Korsakoff syndrome may occur in the general population. However, for some as yet unexplained reason(s) this variant seems to occur more frequently among familial chronic alcoholic men and their male offspring without any history of alcoholism. The inheritance pattern of this enzyme variant as revealed from an Amish pedigree study may be autosomal recessive as previously suggested.

MRI Brain Volume Measurements in Infantile Neuronal Ceroid Lipofuscinosis.

BACKGROUND AND PURPOSE: Infantile neuronal ceroid lipofuscinosis is a devastating neurodegenerative storage disease caused by palmitoyl-protein thioesterase 1 deficiency, which impairs degradation of palmitoylated proteins (constituents of ceroid) by lysosomal hydrolases. Consequent lysosomal ceroid accumulation leads to neuronal injury, resulting in rapid neurodegeneration and childhood death. As part of a project studying the treatment benefits of a combination of cysteamine bitartrate and N-acetyl cysteine, we made serial measurements of patients' brain volumes with MR imaging. MATERIALS AND METHODS: Ten patients with infantile neuronal ceroid lipofuscinosis participating in a treatment/follow-up study underwent brain MR imaging that included high-resolution T1-weighted images. After manual placement of a mask delineating the surface of the brain, a maximum-likelihood classifier was applied to determine total brain volume, further subdivided as cerebrum, cerebellum, brain stem, and thalamus. Patients' brain volumes were compared with those of a healthy population. RESULTS: Major subdivisions of the brain followed similar trajectories with different timing. The cerebrum demonstrated early, rapid volume loss and may never have been normal postnatally. The thalamus dropped out of the normal range around 6 months of age; the cerebellum, around 2 years of age; and the brain stem, around 3 years of age. CONCLUSIONS: Rapid cerebral volume loss was expected on the basis of previous qualitative reports. Because our study did not include a nontreatment arm and because progression of brain volumes in infantile neuronal ceroid lipofuscinosis has not been previously quantified, we could not determine whether our intervention had a beneficial effect on brain volumes. However, the level of quantitative detail in this study allows it to serve as a reference for evaluation of future therapeutic interventions.

Transcriptional regulation of cyclooxygenase-2 gene expression: novel effects of nonsteroidal anti-inflammatory drugs.

Cyclooxygenase-2 (COX-2) gene overexpression is suggested to play important roles in colorectal tumorigenesis. Epidemiological studies revealed that nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and sulindac, which inhibit COX activity, reduce colorectal cancer mortality. Current investigations have focused on delineating the molecular mechanisms that regulate COX-2 gene expression and the roles of NSAIDs in cancer chemoprevention. COX-2 catalyzes the production of prostaglandins (PGs) from arachidonic acid (AA), generated by phospholipases A2 (PLA2s), a family of acyl esterases that cause the release of AA from cellular phospholipids. Pancreatic secretory PLA2 (sPLA2), via its receptor (sPLA2R), transcriptionally activates COX-2 gene expression in several cell types, although a specific transcription factor mediating COX-2 expression has not yet been identified. Here, we report that a transcription factor, CCAAT/enhancer-binding protein beta(C/EBPbeta), plays a critical role in sPLA2IB-induced, receptor-mediated COX-2 gene expression in MC3T3E1 and NIH3T3 cells. Furthermore, treatment of these cells with NSAIDs in the presence of sPLA2IB appears to potentiate the stimulatory effects on COX-2 mRNA and COX-2 protein expression and a concomitant elevation in PG production. Most significantly, NSAID treatment appears to drastically suppress the production of cytosolic PLA2 (cPLA2) mRNA. The lack of sPLA2IB, sPLA2IIA, and sPLA2V mRNA expression in both NIH3T3 and MC3T3E1 cells suggests that cPLA2 is the most likely enzyme that catalyzes the release of AA, the rate-limiting substrate of COX for the production of PGs. Our results suggest that: (a) sPLA2IB receptor-mediated COX-2 expression is mediated via C/EBPbeta; (b) NSAIDs in the presence of sPLA2IB potentiate the stimulatory effects of sPLA2IB on COX-2 mRNA expression; and (c) despite the apparent stimulation of COX-2 expression by NSAIDs, they strikingly deprive COX-2 of its substrate, AA, by suppressing cPLA2 mRNA expression. Both AA and PGs regulate many vital biological functions (e.g., motility and invasiveness) that are dysregulated in most cancer cells, and they have profound effects on cellular differentiation. Our results raise the possibility that deprivation of COX-2 of its substrate by the suppression of cPLA2 mRNA expression is an additional mechanism used by NSAIDs to inhibit tumorigenesis.

Recombinant human uteroglobin inhibits the in vitro invasiveness of human metastatic prostate tumor cells and the release of arachidonic acid stimulated by fibroblast-conditioned medium.

Uteroglobin (UG) is a potent immunomodulatory and antiinflammatory secretory protein with high levels detected in human prostate tissue. We used three human prostate cancer cell lines (DU-145, PC3-M, and LNCaP) to test the hypothesis that UG may modulate invasiveness of prostatic carcinoma cells in the Boyden chamber assay for invasion through a reconstituted basement membrane preparation. Fibroblast-conditioned medium was used as the chemoattractant. The most invasive cell line was DU-145, followed by PC3-M, whereas the androgen-dependent LNCaP cell line exhibited extremely low invasive potential. Pretreatment of DU-145 and PC3-M cells for 24 h with 0.01, 0.1, or 1.0 microM recombinant UG had no effect on basal invasiveness but inhibited fibroblast-conditioned medium-stimulated invasion in a dose-dependent manner, reaching up to 60.2 and 87.9% inhibition of DU-145 and PC3-M, respectively. UG had no effect on either cell-reconstituted basement membrane adhesion or simple chemotaxis in the absence of reconstituted basement membrane. UG also strongly inhibited the biphasic release of [14C]-labeled arachidonic acid from fibroblast-conditioned medium-stimulated DU-145 cells. These results suggest that UG may modulate prostate tumor cell invasiveness and that the mechanism may include inhibition of the arachidonic acid signal cascade.

Expression of antisense osteopontin RNA inhibits tumor promoter-induced neoplastic transformation of mouse JB6 epidermal cells.

Elevated expression of osteopontin (OPN), a secreted adhesive phosphoglycoprotein, is frequently associated with many transformed cell lines of epithelial and stromal origin. Moreover, several clonal lines of preneoplastic JB6 cells derived from Balb/c mouse epidermal cultures (Colburn et al., 1978, 1979), upon treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA), become irreversibly oncogenic and concomitantly synthesize OPN at elevated levels (Smith and Denhardt, 1989). In the present study we sought to determine whether OPN expression facilitates transformation of such preneoplastic (initiated) cells. We transfected TPA-promotable JB6 c141.5a cells with an expression vector containing mouse OPN cDNA in antisense orientation under transcriptional control of dexamethasone-inducible MMTV-LTR promoter. Four stably transfected clones, which expressed drastically reduced levels of OPN in the presence of both dexamethasone and TPA, were characterized. We found that (a) more than 20 copies of OPN antisense cDNA were stably incorporated into the genome of cells from two of these clones that were examined by Southern blot analysis; (b) dexamethasone-induced expression of antisense OPN RNA prevented augmented OPN expression at both mRNA and protein levels following TPA treatment; and (c) cells from all four clones failed to form colonies in soft agar medium containing both dexamethasone and TPA. Taken together, these data demonstrate that inhibition of elevated OPN expression blocks TPA-induced anchorage-independent growth of JB6 c141.5a cells, suggesting the possibility that OPN overproduction is causally related to transformation of preneoplastic cells.

Allelic loss and somatic differentiation in human male germ cell tumors.

The complex but poorly understood human male germ cell tumors offer unusual opportunities for the genetic analysis of malignant transformation and embryonal differentiation in a pluripotential stem cell lineage. Histologically, these tumors are divided into two major subgroups, seminomas which are characterized by inability to express embryonal differentiation, and non-seminomas which are characterized by ability to express embryonal as well as extra-embryonal patterns of differentiation. To understand the role of genetic factors in the development of these tumors and the regulation of differentiation expressed by them, we carried out a detailed allelotype analysis by the loss of heterozygosity assay. This analysis revealed frequent deletions in known tumor suppressor genes (RB1, DCC, NME), a number of previously described sites of candidate tumor suppressor genes (3p, 9p, 9q, 10q, 11p, 11q and 17p), as well as several novel sites (2p, 3q, 5p, 12q, 18p and 20p). Our results also showed that well differentiated teratomas exhibit a significantly higher level of allelic loss compared to the less differentiated embryonal carcinomas. In addition, certain loci and genes exhibited frequent non-random deletion in teratomas (D3S32, D3S42, D5S12, D10S25, D11S12, RB1, TP53, NME1, NME2, D17S4, D18S6 and D20S6) and embryonal carcinomas (IFNB, D9S27). Among these loci, the NME genes were notable for a high degree of genetic loss (> 70%) in teratomas. These results suggested that nonrandom loss or inactivation of certain genes may be associated with tumor development and loss or inactivation of other genes may be associated with somatic differentiation.

Cells in vivo and in vitro from osteopetrotic mice homozygous for c-src disruption show suppression of synthesis of osteopontin, a multifunctional extracellular matrix protein.

Mice carrying homozygous disruption of the c-src proto-oncogene (Src-/-) develop osteopetrosis due to an impaired ability of osteoclasts to adhere to the bone surface and/or to form bone-resorbing ruffled border. It has also been reported that osteopontin (OPN), a secreted phosphoprotein, mediates osteoclast adherence to the bone matrix. We report here that cells from Src-/- mice, both in vitro and in vivo, express OPN mRNA and protein at a significantly reduced level as compared to cells from Src+/- and +/+ animals, suggesting a potential role for the proto-oncogene c-src in the regulation of OPN gene expression. Our data also show that OPN gene expression can be induced by treatment of SR-/- cells with epidermal growth factor (EGF) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Results obtained from studies using inhibitors of receptor tyrosine kinases (RTKs) and protein kinase C (PKC) suggest that PKC and RTK are positioned in a pathway with PKC as the downstream effector for the EGF-induced OPN gene expression in SRC-/- cells, and that pp60c-src and EGF may regulate OPN gene expression through a common signalling pathway. Furthermore, contrary to published reports, our study shows that EGF-mediated cell signalling does not require functional interaction between the EGF-receptor and pp60c-src.

Partial characterization of a uteroglobin-like protein in the human uterus and its temporal relationship to prostaglandin levels in this organ.

During the past decade several corticosteroid-dependent, low mol wt proteins with phospholipase-A2 (PLA2) inhibitory activity have been described. This family of proteins is collectively known as lipocortins. Blastokinin or uteroglobin (utg), a progesterone-induced protein, first discovered in the pregnant rabbit uterus, is also a potent PLA2 inhibitor, but genetically distinct from lipocortins. Although utg has been found in rabbits, its presence in humans has not been well established. Here, we present biochemical, immunological, and immunohistological evidence for the detection of a utg-like protein in the human uterus. Since inhibition of PLA2 may modulate tissue eicosanoid levels and since rabbit utg has been reported to be a potent PLA2 inhibitor, we also studied the temporal relationship between utg and tissue prostaglandin E2 and F2 alpha levels in estrogen- and progesterone-dominated endometrial tissue. We found an inverse temporal relationship between utg-like protein and eicosanoid levels in this organ. Since some eicosanoids (e.g. prostaglandins, leukotrienes, etc.) are known to be involved in smooth muscle contractility and inflammatory processes, our findings may help to understand the pathogenesis of some human disorders in which abnormal eicosanoid production occurs.

Palmitoyl-protein thioesterase gene expression in the developing mouse brain and retina: implications for early loss of vision in infantile neuronal ceroid lipofuscinosis.

Mutations in the palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL), the clinical manifestations of which include the early loss of vision followed by deterioration of brain functions. To gain insight into the temporal onset of these clinical manifestations, we isolated and characterized a murine PPT (mPPT)-cDNA, mapped the gene on distal chromosome 4, and studied its expression in the eye and in the brain during development. Our results show that both cDNA and protein sequences of the murine and human PPTs are virtually identical and that the mPPT expression in the retina and in the brain is temporally regulated during development. Furthermore, the retinal expression of mPPT occurs much earlier and at a higher level than in the brain at all developmental stages investigated. Since many retinal and brain proteins are highly palmitoylated and depalmitoylation by PPT is essential for their effective recycling in the lysosomes, our results raise the possibility that inactivating mutations of the PPT gene, as occur in INCL, are likely to cause cellular accumulation of lipid-modified proteins in the retina earlier than in the brain. Consequently, the loss of vision occurs before the deterioration of brain functions in this disease.

Sequence of centromere separation of mitotic chromosomes during human cellular aging.

The sequence of centromere separation of mitotic chromosomes derived from lymphocytes of 17 individuals of various ages was studied. A comparison of the mean CSI (centromeric separation index) values for individual chromosomes in the complement indicated a significant difference between the older and the younger subjects only with regard to the chromosome nos. 6, 11, and 19 in both sexes and the X chromosome exclusively in the female. With age, chromosome no. 6 in both males and females and the X chromosome in females showed premature centromeric separation whereas chromosome nos. 11 and 19 in both males and females displayed delayed centromeric separation. Our study, therefore, indicates that changes in the sequence of centromeric separation of certain chromosomes do occur in lymphocytes during chronological aging in humans.