Extracellular domain dependence of PTPalpha transforming activity.

Two isoforms of the transmembrane protein tyrosine phosphatase PTPalpha, which differ by nine amino acids in their extracellular regions, are expressed in a tissue-specific manner. Over-expression of the shorter isoform transforms rodent cells, and it has previously been reasonable to assume that this was a direct consequence of its dephosphorylation and activation of Src. Transformation by the longer wild-type isoform has not previously been studied. We tested the activities of both isoforms in NIH3T3 cells and found that, while both dephosphorylated and activated Src similarly, only the shorter isoform induced focus formation or anchorage-independent growth. Differences in phosphorylation of PTPalpha at its known regulatory sites, Grb2 binding to PTPalpha, phosphorylation level of focal adhesion kinase by PTPalpha, or overall localization were excluded as possible explanations for the differences in transforming activities. The results suggest that transformation by PTPalpha involves at least one function other than, or in addition to, its activation of Src and that this depends on PTPalpha's extracellular domain. Previous studies have suggested that PTPalpha might be a useful target in breast and colon cancer therapy, and the results presented here suggest that it may be advantageous to develop isoform-specific therapeutic reagents.

Foxg1 haploinsufficiency reduces the population of cortical intermediate progenitor cells: effect of increased p21 expression.

Foxg1 is a transcription factor that is critical for forebrain development. Foxg1(+/Cre) mice were used to test the hypotheses 1) that the subventricular zone (SZ) generates supragranular neurons, 2) that Foxg1-regulated activities define the output from the SZ, and 3) that Foxg1 is involved in the suppression of p21-initiated cell-cycle exit. Foxg1(+/Cre) mice have thinner neocortices than wild-type controls, specifically in the supragranular layers, as detected by Brn2 immunostaining. Cell proliferation in the ventricular zone (VZ) and SZ was examined to investigate the reduction in upper layer neurons. The number of cycling VZ cells was similar in Foxg1(+/+) and Foxg1(+/Cre) brains. Interestingly, cell proliferation in the SZ and intermediate progenitor cell (IPC) production (noted by Tbr2-immunostaining) was reduced in Foxg1(+/Cre) brains. These decreases coincided with increased expression of the cell-cycle inhibitor p21 in the VZ and SZ. Furthermore, colocalization of p21 with markers of cell proliferation and IPCs indicated that p21 was temporally expressed to influence the proliferative fate of IPCs. Thus, the present data are consistent with the above hypotheses, particularly, that during corticogenesis, Foxg1-regulated activities enable the expansion of the IPC population likely through suppression of p21-dependent cell-cycle exit.

Generation and use of primary rat cultures for studies of the effects of ethanol.

In vivo studies are ideal for identifying the phenomenology of ethanol toxicity and teratology. They are limited in being able to explore cellular and molecular mechanisms of action. Two types of culture models have proven to be very instructive: monolayer primary cultures of dissociated cells and organotypic slice cultures. Dissociated cell preparations have the advantage of being enriched populations of cells, whereas the organotypic cultures have the advantage of providing normal cell associations. Details for the methods used to generate these preparations are described. As ethanol is a volatile liquid, the success of a culture model depends upon stabilizing the ethanol content in the culture medium. A method to maintain the ethanol concentration is described.

Nuclear calpain regulates Ca2+-dependent signaling via proteolysis of nuclear Ca2+/calmodulin-dependent protein kinase type IV in cultured neurons.

Accumulating evidence indicates that calpains can reside in or translocate to the cell nucleus, but their functions in this compartment remain poorly understood. Dissociated cultures of cerebellar granule cells (GCs) demonstrate improved long-term survival when their growth medium is supplemented with depolarizing agents that stimulate Ca(2+) influx and activate calmodulin-dependent signaling cascades, notably 20 mm KCl. We previously observed Ca(2+)-dependent down-regulation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) type IV, which was attenuated by calpain inhibitors, in GCs supplemented with 20 mm KCl (Tremper-Wells, B., Mathur, A., Beaman-Hall, C. M., and Vallano, M. L. (2002) J. Neurochem. 81, 314-324). CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival. Here, we demonstrate by immunolocalization/confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mm KCl. Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and non-nuclear calpain substrates were not degraded under chronic depolarizing culture conditions. Depolarization and Ca(2+)-dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca(2+)-CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by approximately 10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced. We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca(2+)-CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mm KCl. This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca(2+) influx.

Trophic agents that prevent neuronal apoptosis activate calpain and down-regulate CaMKIV.

CaMKIV is enriched in neuronal nuclei and mediates Ca2+-dependent survival via transcription factor phosphorylation. Cultured cerebellar granule neurons were used to examine whether distinct modes of Ca2+ signaling differentially modulate CaMKIV expression and function. For long-term survival, these neurons require 25 mm KCl or NMDA, which stimulates Ca2+ entry through voltage-sensitive Ca2+ channels or NMDA receptors (NRs). Lower levels of Ca2+ entry through NRs support survival of a neuronal subpopulation grown in 5 mm KCl media. Several effects were demonstrated: (i) sustained exposure to 25 mM KCl or 140 microM NMDA produced CaMKIV down-regulation, compared to 5 mM KCl cultures; (ii) CaMKIV down-regulation was attenuated by nifedipine, APV and CaM kinase inhibitors, indicating that it is Ca2+ dependent and reversible; (iii) down-regulation was both selective for nuclear substrates and calpain-mediated; (iv) proteolysis was exacerbated by leptomycin B, a nuclear export inhibitor. Although CaMKIV proteolysis by trophic agents seems paradoxical in light of evidence supporting its critical role in survival, the CaMKIV/CREB signal transduction pathway was preserved, as assessed by CaM kinase-mediated CREB phosphorylation, and the ability of CaM kinase inhibitors to interfere with KCl-mediated survival. We hypothesize that limited calpain-mediated proteolysis of CaMKIV is a negative feedback response to the sustained activation of a Ca2+ and CaMKIV signaling pathway by these agents.