HAI-2 suppresses the invasive growth and metastasis of prostate cancer through regulation of matriptase.

Dysregulation of cell surface proteolysis has been strongly implicated in tumorigenicity and metastasis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer (PCa) cell migration, invasion, tumorigenicity and metastasis using a human PCa progression model (103E, N1, and N2 cells) and xenograft models. N1 and N2 cells were established through serial intraprostatic propagation of 103E human PCa cells and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was revealed to gradually increase throughout the serial isolations (103E

Upregulation of the HLH Id gene family in neural progenitors and glial cells of the rat spinal cord following contusion injury.

Spinal cord injury (SCI) leads to a complex sequence of cellular responses, including astrocyte activation, oligodendrocyte death, and ependymal cell proliferation. Inhibitors of DNA binding (Id1, Id2, Id3) belong to a helix-loop-helix (HLH) gene family. Id genes have been implicated in playing a vital role in the proliferation of many cell types, including astrocytes and myoblasts. In the present study, the expression of Id family members in spinal cord after contusion injury was investigated by in situ hybridization. Id1, Id2, and Id3 mRNA expression was upregulated 5 mm rostral and caudal to the lesion center, and reached maximal levels 3 days after SCI. In addition, cell populations expressing Id1, Id2, and Id3 mRNA were maximally increased 3 days after SCI. The increase in Id2 and Id3 mRNA expression and Id2 and Id3 mRNA+ cells was still observed at 8 days. The Id mRNA expressing cells were phenotyped by combining immunostaining of cell-specific markers with in situ hybridization. Glial fibrillary acidic protein (GFAP)+ astrocytes were found to express all three Id mRNA, whereas S-100alpha+ astrocytes only expressed high levels of Id2 and Id3 mRNA. Cells having a neural progenitor morphology and the marker nestin appeared after SCI and they expressed Id1, Id2, and Id3 mRNA. Interestingly, some Rip+ oligodendrocytes located in the areas close to the central canal expressed Id3 mRNA after injury. In conclusion, Id genes are upregulated in a time-dependent manner in astrocytes, oligodendrocytes, and neural progenitor subpopulations after SCI, suggesting that they play major roles in cellular responses following SCI.

Expression of neural cell adhesion molecule in spinal cords following a complete transection.

Neural cell adhesion molecule (NCAM) regulates tissue organization during development and in the adult. NCAM upregulation occurs after an injury to brains and sciatic nerves. However, little is known about NCAM expression after spinal cord injury (SCI). By using a complete spinal cord transection with a 5 mm tissue removal, an increase in the NCAM level is detected in spinal cord stumps proximal and distal to the transection site at 1 d and 3 d post injury, while its expression at 8 d is declined to a lower level than that observed in sham-operated spinal cords. The strong NCAM expression is present in motor neurons at 3 d post transection whereas the intensive NCAM immunostaining is localized in dorsal sensory and corticospinal fiber tracts at 8 d following injury. Collectively, NCAM level is elevated and strongly expressed in dorsal fiber tracts after SCI, implying that the endogenous process for spinal cord regeneration may take place after SCI.

Tumor necrosis factor-alpha modulates the proliferation of neural progenitors in the subventricular/ventricular zone of adult rat brain.

Little is known about the response of neural progenitors to inflammation following injuries of the central nervous system. In combination with bromodeoxyuridine (BrdU) intraperitoneally (i.p.) injected, tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine that increased ED1+ activated microglia/macrophage population at injured sites, was administrated into adult rat brains. No difference in the immunostaining for proliferating cell nuclear antigen (PCNA) was observed in the subventricular/ventricular zone (SVZ/VZ) between TNF-alpha injected sites and controls. However, BrdU+ cells were apparently observed in the SVZ/VZ proximal to TNF-alpha injected site, and the number of BrdU+ cells increased at 6 and 24 h post injection. Since cell apoptosis was rarely found in the SVZ/VZ after TNF-alpha injection, these observations suggest that the diffusible TNF-alpha may directly and/or indirectly modulate the proliferation of neural progenitors.

Myelin basic protein (MBP) and MBP peptides are mitogens for cultured astrocytes.

After CNS demyelination, astrogliosis interferes with axonal regeneration and remyelination. We now provide evidence that myelin basic protein (MBP) can contribute to this observed astrocyte proliferation. We found that astrocytes grown in either serum-containing or serum-free medium proliferate in response to MBP. The mitogenic regions of MBP in both media were MBP(1-44), MBP(88-151) and MBP(152-167). The mitogenic effect of these individual peptides was potentiated by simultaneous treatment with microglia conditioned media (CM). MBP-induced proliferation was inhibited by suramin at concentrations known to block the fibroblast growth factor receptor (FGFR), whereas neither MBP(1-44), MBP(88-151) nor MBP(152-167) were affected. Cholera toxin B, that binds to ganglioside GM(1), inhibited the mitogenicity of MBP(1-44) and had no significant effect on the mitogenicity of MBP, MBP(88-151) or MBP(152-167). Treatment of astrocytes with MBP and MBP(152-167) caused a modest and transitory elevation of intracellular calcium, whereas treatment with MBP(1-44) resulted in a substantial and sustained increase in intracellular calcium. These results suggest that for cultured astrocytes 1) FGFR and extracellular calcium play a major role in MBP mitogenicity; 2) MBP(1-44), MBP(88-151) and MBP(152-167) are the mitogenic regions of MBP; 3) MBP(1-44) and MBP(152-167) interact with ganglioside GM(1) and FGFR, respectively; 4) Component(s) present in microglial CM potentiate the mitogenicity of MBP(1-44), MBP(88-151) and MBP(152-167). These data support the hypothesis that MBP related peptides in conjunction with microglial secreted factors may stimulate astrogliosis after demyelination in vivo.

Responses of microglia and neural progenitors to mechanical brain injury.

Neural cells have distinct responses to CNS injury; however, neural progenitor response to CNS injury is not yet documented. Stab injury combined with injection of bromodeoxyuridine (BrdU), a thymidine analog, into adult rat cortex above the lateral ventricle for 10 min resulted in activated microglia/macrophage infiltration along with nuclear factor kappa B (NF kappa B)/p65 activation at the lesion site. Most NF kappa B/p65+ cells displayed a phagocytic morphology. Under these conditions, profound cell apoptosis took place in the injured corpus callosum, but not in the subventricular/ventricular zone (SVZ/VZ). The SVZ/VZ-derived neural progenitors in both injected and non-injected contralateral hemispheres showed strong BrdU immunostaining, indicating that SVZ/VZ-derived neural progenitors of both hemispheres may undergo DNA synthesis in response to unilateral brain injury.

Tumor necrosis factor-alpha regulation of the Id gene family in astrocytes and microglia during CNS inflammatory injury.

The inhibitors of DNA binding (Id) gene family is highly expressed during embryogenesis and throughout adulthood in the rat central nervous system (CNS). In vitro studies suggest that the Id gene family is involved in the regulation of cell proliferation and differentiation. Recently, Id gene expression was shown to be expressed in immature and mature astrocytes during development and upregulated in reactive astrocytes after spinal cord injury. These results suggest that the Id gene family may play an important role in regulating astrocyte development and reactivity; however, the factors regulating Id expression in astrocytes remain undefined. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, is thought to play a crucial role in astrocyte/microglia activation after injury to the CNS. To determine if TNF alpha plays a role in Id gene expression, we exogenously administered TNF alpha into developing postnatal rats. We report that TNF alpha injections resulted in a rapid and transient increase in both cell number and mRNA expression for Id2 and Id3 when compared to levels observed in noninjected or control-injected animals. Id1 mRNA levels were also upregulated after TNF alpha treatment, but to a lesser degree. Significant increases in TNF alpha-induced Id2 and Id3 mRNA were observed in the ventricular/subventricular zone, cingulum and corpus callosum. TNF alpha also increased Id2 mRNA expression in the caudate putamen and hippocampus at the injection site. Id2 and Id3 mRNA+ cells were identified as GFAP+ and S100 alpha + astrocytes as well as ED1+ microglia. This is the first report to show TNF-alpha-induced modulation of the Id gene family and suggests that Id may be involved in the formation of reactive astrocytes and activated microglia in the rodent brain. These results suggest a putative role for the Id family in the molecular mechanisms regulating cellular responsiveness to TNF alpha and CNS inflammation.

Myelin basic protein and myelin basic protein peptides induce the proliferation of Schwann cells via ganglioside GM1 and the FGF receptor.

Myelin basic protein (MBP) and two peptides derived from MBP (MBP(1-44) and MBP(152-167)) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of 125I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of 125I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP(1-44)) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP(1-44) and MBP(152-167) associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.

Id1, Id2, and Id3 gene expression in neural cells during development.

Id1, Id2, and Id3 mRNA are expressed mainly in the proliferating ependymal cell zone of the mouse brain during embryogenesis. In this study, the expression pattern and cell phenotypes of the Id family mRNA were examined in postnatal and adult rat brain. The expression of Idl and Id3 mRNA in rat brain was observed in the cortex layer 1, corpus callosum, ventricular/subventricular zone (VZ/ SVZ), and the CA1-4 layers of the hippocampus at postnatal day 1 (P1) through P14, whereby it declined at 2 months. In general, the developmental pattern of Idl mRNA coincided with the pattern observed for Id3 mRNA. Similar to Id1 and Id3, Id2 mRNA was highly expressed in the corpus callosum, VZ/SVZ, and the hippocampus. Examination of Id2 mRNA revealed high levels in the cortex and caudate putamen at P1 through P14, whereas a decline was observed in its expression in the adult cortex. In P5 rat cerebellum, all Id mRNA examined were found in the internal granular cell layers; however, at this time point, only Id2 mRNA expression was detected in the differentiating zone of the external granular cell layers, preferentially localizing to adult Purkinje cells. Furthermore, only Id2 mRNA expression in brain was observed in NF+ neurons at P5. Examination of S100alpha+ and GFAP+ astrocytes, revealed the presence of all three mRNAs, whereas the expression of Id2 and Id3 mRNA was absent in 04+ immature oligodendrocytes. These data suggest that the spatial and temporal kinetic patterns during development, as well as cellular specificity, of the Id gene family may play a critical role in neural precursor cell proliferation and cell divergence.

Expression and functional role of the Id HLH family in cultured astrocytes.

The Id family of helix-loop-helix factors (Id1, Id2, and Id3) expressed in many types of cells has been reported to negatively regulate myoblast differentiation and is required for G1/S progression of arrested fibroblasts. Our previous studies have indicated that Id1, Id2, and Id3 mRNA expression appear in the subventricular zone of 1-day-old rat brains. At later ages, Id3 mRNA was only expressed in astrocytes. We now report that Id1 and Id3 mRNA expression increased in astrocytes during the first hour of serum stimulation. Subsequently, the Id1 and Id3 mRNA levels gradually declined to basal level as observed in cultures without serum stimulation. However, there was no significant difference in Id2 mRNA expression between serum-treated and control astrocyte cultures within 1 h of serum induction. In addition, a strong nuclear immunostaining for Id2 and Id3 proteins was observed 24 h after serum stimulation. This observation is consistent with our results that show an increase in Id2 and Id3 protein levels following 24 h serum induction. Furthermore, DNA synthesis in FCS-stimulated astrocytes was blocked by antisense oligonucleotides against Id3 mRNA. The addition of Id3 antisense oligonucleotides caused approximately 50% reduction in Id3 mRNA and protein levels when compared to that in sense-treated cultures. The results indicate that the inhibition of DNA synthesis in FCS-stimulated astrocytes is due to a decrease in Id3 levels by the antisense. These observations suggest that Id3 may play an important role in the regulation of astrocyte proliferation.

Two mitogenic regions of myelin basic protein interact with different receptors to induce Schwann cell proliferation in a cAMP dependent process.

Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one mitogenic region within the first 44 residues of the aminoterminus (1-44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152-167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of 125I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-beta nor PDGF-BB displaced cell surface bound 125I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP interacted with ganglioside GM1, whereas MBP did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP, while the FGF receptor mediates the mitogenic effect of MBP. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens.

Exogenous myelin basic protein promotes oligodendrocyte death via increased calcium influx.

Treatment of cultured oligodendrocytes (OLGs) with micromolar quantities of myelin basic protein (MBP) caused a rapid, MBP-dose-dependent cell death. In contrast, a 72-hr incubation of OLGs with MBP peptides (1-44, 47-87, 88-151, or 152-167) at comparable concentrations had no effect on cell viability. MBP and MBP peptides (1-44 and 88-151) have been shown to interact with ganglioside GM1 (Tzeng et al.: J Neurochem Res: 42:758-767, 1995). This interaction has been reported to increase calcium influx. Therefore, using the fluorescent dye Indo-1 and an ACAS laser cytometer, we examined the level of intracellular calcium in OLGs after MBP treatment. MBP was shown to provoke a rapid, dramatic, and sustained rise of intracellular calcium in most OLGs. The levels of elevated intracellular calcium were sustained and did not return to baseline even after 10 min. This increase of intracellular calcium was suppressed in the presence of EGTA, indicating that the [Ca2+]i rise was due to the entry of extracellular calcium. Incubation of cultured OLGs with MBP peptides (1-44 or 88-151) caused a modest and transitory elevation of intracellular calcium ions in a lower percentage of OLGs. The potent OLG cytotoxicity of intact MBP and the loss of potency after proteolysis raise the possibility that MBP proteolysis during demyelination protects OLGs from death.

Norepinephrine-induced contractile responses in isolated rat aortae from different duration of diabetes.

The present study examined contractile responses to norepinephrine in isolated aortae from 4-, 8-, and 12-week duration of streptozocin-induced diabetic rats. The pD2 value and the maximum contractile responses were significantly increased in 8-week and 12-week diabetic aortae, respectively, compared to the age-matched control aortae. Moreover, the bethanechol-induced relaxation of 12-week diabetic aortae preconstricted with norepinephrine was also significantly different from the age-matched control aortae. On the other hand, the KCl-, phenylephrine-induced contraction in 4-, 8-, and 12-week diabetic and age-matched control aortae were similar. Presumably the duration of diabetes altered the sensitivity and responsiveness of aortae to norepinephrine. The possibility of the enhanced norepinephrine response in diabetic aortae was discussed.

Post-treatments with sodium arsenite during G2 enhance the frequency of chromosomal aberrations induced by S-dependent clastogens.

Treatment with sodium arsenite during the G2 phase potentiated the chromatid breaks and chromatid exchanges induced by ultraviolet light or 4-nitroquinoline 1-oxide but not those induced by methyl methanesulfonate, ethyl methanesulfonate, mitomycin C or cisplatin in Chinese hamster ovary cells. A comparison was made between the effects of treatment during G2 with sodium arsenite, cytosine-beta-D-arabinofuranoside, aphidicolin, hydroxyurea, caffeine, 3-aminobenzamide and novobiocin on the frequency of chromosomal aberrations induced by the above-mentioned S-dependent clastogens. It was found that the effects varied considerably, both quantitatively and qualitatively. However, potentiation was more often observed in the chromosomal aberrations induced by ultraviolet light and 4-nitroquinoline 1-oxide than by other S-dependent clastogens, and the frequency of chromatid exchanges was potentiated only in cells pretreated with ultraviolet light or 4-nitroquinoline 1-oxide. Furthermore, for all of the S-dependent clastogens studied, treatment with cytosine-beta-D-arabinofuranoside during the G2 phase potentiated the frequency of chromatid breaks but not the frequency of chromatid exchanges.

Pyrimidine metabolism in Tritrichomonas foetus.

The anaerobic parasitic protozoa Tritrichomonas foetus is found incapable of de novo pyrimidine biosynthesis by its failure to incorporate bicarbonate, aspartate, or orotate into pyrimidine nucleotides or nucleic acids. Uracil phosphoribosyltransferase in the cytoplasm provides the major pyrimidine salvage for the parasite. Exogenous uridine and cytidine are mostly converted to uracil by uridine phosphorylase and cytidine deaminase in T. foetus prior to incorporation. T. foetus cannot incorporate labels from exogenous uracil or uridine into DNA; it has no detectable dihydrofolate reductase or thymidylate synthetase and is resistant to methotrexate, pyrimethamine, trimethoprim, and 5-bromovinyldeoxyuridine at millimolar concentrations. It has an enzyme thymidine phosphotransferase in cellular fraction pelleting at 100,000 X g that can convert exogenous thymidine to TMP via a phosphate donor such as p-nitrophenyl phosphate or nucleoside 5'-monophosphate. Thymidine salvage in T. foetus is thus totally dissociated from other pyrimidine salvage.

Factor 420-dependent pyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.