Stanniocalcin 1 is important for poststroke functionality, but dispensable for ischemic tolerance.

Stanniocalcin 1 (STC1), originally described as an antihypercalcemic hormone in fish, is highly expressed in differentiated mammalian neurons. Mild hypoxic treatment and focal cerebral ischemia induce upregulation of STC1 in the brain. These findings prompted us to investigate whether STC1 contributes to neuroprotection after ischemia and whether STC1 is required for development of ischemic tolerance. We induced 60 minutes of temporary middle cerebral artery occlusion in wild type (WT) and STC1-deficient mice (STC1(-/-)) with or without prior hypoxic preconditioning (HPC, 8% oxygen for 6 hours followed by reoxygenation for 24 hours). Infarct sizes, neurological scores, and Stc1, Stc2, and Il-6 mRNA brain levels were measured 24 hours after ischemia. Additionally, we examined blood-brain barrier (BBB) integrity (Evans Blue fluorescence) under normal conditions and 0 and 24 hours after hypoxia. STC1(-/-) and WT mice developed brain infarcts of similar size. In both strains, HPC triggered ischemic tolerance with similar reduction in infarct size. However, STC1(-/-) mice had worse neurological scores in both scenarios. HPC induced upregulation of STC1 and STC2 in WT mice and of STC2 in STC1(-/-) mice. Ischemic STC1(-/-) mice showed significantly lower Il-6 mRNA expression than ischemic WT mice. Evans Blue fluorescence levels showed no difference in between WT and STC1(-/-) mice under evaluated conditions, thus BBB integrity is preserved despite STC1 deficiency. STC1 was not crucial for the development of ischemic tolerance triggered by HPC or for preserving BBB integrity but may be involved in functional recovery after stroke.

Expression of stanniocalcin in the epithelium of human choroid plexus.

Stanniocalcin (STC) is a 28 kD glycoprotein hormone originally found in bony fish in which it regulates calcium/phosphate homeostasis and protects against hypercalcemia. The recently characterized mammalian STC shows about 70% homology with fish STC. The epithelial cells of proximal tubuli in human and rat kidney and brain neurons have been found to express STC. Here we show that the epithelium of the choroid plexus, already at 16 weeks of fetal age, and of plexus papillomas, synthesize and express STC. Our findings suggest that STC may be of importance for the distribution of calcium and phosphate between the cerebrospinal fluid and blood.

Regulation of neural differentiation by normal and mutant (G654A, amyloidogenic) gelsolin.

Gelsolin belongs to a family of proteins that modulate the structural dynamics of cytoskeletal actin. Gelsolin activity is required for the redistribution of actin occurring during membrane ruffling, cell crawling, and platelet activation. A point mutation (G654A) in the gelsolin gene causes a dominantly inherited systemic amyloidosis called familial amyloidosis of the Finnish type (FAF). This disease is characterized by a cranial neuropathy that cannot be explained solely by amyloid deposits. To address the question of whether gelsolin has a specific role in neural cell development, we transfected cDNA for wild type and G654A point-mutated gelsolin into a neural cell line, Paju, which can be induced to differentiate by treatment with phorbol 12-myristate 13-acetate. Overexpressed wild type gelsolin inhibited neural differentiation whereas mutated gelsolin did not, indicating that appropriate gelsolin activity is essential for neural sprouting. The G654A mutant gelsolin induced stabilization of F-actin and reduced the plasticity of neural development. This provides a novel etiopathogenetic mechanism for the neuronal dysfunction in FAF.

High expression of stanniocalcin in differentiated brain neurons.

Stanniocalcin (STC) is a glycoprotein hormone first found in fish, in which it regulates calcium homeostasis and protects against hypercalcemia. Human and mouse stc cDNA were recently cloned. We found a dramatically upregulated expression of STC during induced neural differentiation in a human neural crest-derived cell line, Paju. Immunohistochemical staining of sections from human and adult mouse brain revealed abundant presence of STC in the neurons with no activity in the glial cells. STC expression was not seen in immature brain neurons of fetal or newborn mice. Given that STC has been found to regulate calcium/phosphate metabolism in some mammalian epithelia, we suggest that STC may act as a regulator of calcium homeostasis in terminally differentiated brain neurons.

2-Methoxyestradiol-induced phosphorylation of Bcl-2: uncoupling from JNK/SAPK activation.

The natural estrogen metabolite 2-methoxyestradiol (2ME) is anti-angiogenic in vivo and a strong growth inhibitor in vitro. The growth inhibition is due to mitotic arrest and apoptosis. These effects are reminiscent of those induced by taxol, and appear to be mediated by inhibition of microtubule dynamics. Here we have studied the cellular response to 2ME in regard to potential mediators of the observed cellular changes. 2ME treatment increases the insoluble polymerized fraction of cellular tubulin similar to taxol, and in contrast to the microtubule depolymerizing drugs such as colcemid and vincristine. This stabilization following 2ME treatment is accompanied by phosphorylation and inactivation of Bcl-2 increasing gradually from 2-24 hours. To study the pathway leading to Bcl-2 phosphorylation we analyzed Raf-1 and JNK/SAPK kinases, both of which have been reported to be involved in Bcl-2 inactivation. Our results indicate that Raf-1 is phosphorylated in response to 2ME, but this occurs later than Bcl-2 phosphorylation suggesting that Raf-1 is not directly phosphorylating Bcl-2. JNK/SAPK was activated rapidly after 2ME treatment. However, this activation was transient and returned to undetectable levels by 2 hours of treatment, demonstrating that JNK/SAPK is not directly phosphorylating Bcl-2. Taken together with previous results indicating that overexpression of JNK/SAPK leads to Bcl-2 phosphorylation, our results would support a model where JNK/SAPK is indirectly phosphorylating Bcl-2.

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells.

We studied gene expression in relation to induced neural differentiation in a human neural crest-derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12-myristate 13-acetate was analyzed by differential display reverse transcription PCR. A strongly up-regulated expression of decay-accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement-mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up-regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.

BCL2 regulates neural differentiation.

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.

The gene coding for erythrocyte protein band 7.2b (EPB72) is located in band q34.1 of human chromosome 9.

Human erythrocyte integral membrane protein band 7 (also termed protein 7.2b or stomatin) is involved in the Na+/K+ permeability of red cells. A cDNA clone coding for this protein was used as a probe to determine the chromosomal localization of the gene (EPB72). Southern blot analysis of somatic cell hybrid DNA panels localized the human EPB72 gene to chromosome band 9q34.1.