Localization of pleiotrophin and its mRNA in subpopulations of neurons and their corresponding axonal tracts suggests important roles in neural-glial interactions during development and in maturity.

Trophic factors are being increasingly recognized as important contributors to growth, differentiation, and maintenance of viability within the mammalian nervous system during development. Pleiotrophin (PTN) is a secreted 18-kDa heparin binding protein that stimulates mitogenesis and angiogenesis and neurite and glial process outgrowth guidance activities in vitro. We localized the sites and time course of expression of the Ptn gene and its protein product in developing and adult mouse nervous system. Expression of the Ptn gene was first observed at embryo day 8.5 (E8.5). At E12.5, transcripts of the Ptn gene were localized in developing neuroepithelium at sites of active cell division in the spinal cord and brain. At E15.5, transcripts were found in the somata of some but not all neurons and glia whereas in the adult its pattern of expression was nearly exclusively restricted to the brain. The PTN protein was found almost entirely in association with the axonal tracts during development and in adults. Furthermore, as opposed to the finding of PTN in both central and peripheral nervous systems during development, PTN was not expressed beyond the exit where axonal tracts become the peripheral nervous system in adults. At all sites and times examined, the somata that contained Ptn transcripts corresponded with the axonal tracts that contained the PTN protein. The results establish that Ptn is expressed in early development at sites of active mitogenesis in developing neuroepithelium and later in both glial cells and neurons at sites of neuronal and glial process formation in developing axonal tracts. The findings establish a correspondence in the localization of PTN within the nervous system at sites of normal developmental processes that correlate with the functional activities of PTN previously described in vitro.

WT1, the Wilms' tumor suppressor gene product, represses transcription through an interactive nuclear protein.

The Wilms' tumor suppressor gene, wt1, encodes a transcription factor of the zinc finger family. Mutations in WT1 have been detected in subsets of Wilms' tumor and in patients with the Denys-Drash Syndrome. In order to determine how WT1 regulates transcription and perhaps the consequences that mutations in WT1 may have, we established that residues 85-124 and 181-250 of WT1 constitute domains that function independently with a DNA binding domain to repress or activate transcription, respectively, and function equally effectively with heterologous promoters, suggesting the activator and repressor domains interact with nuclear components of general importance. To seek evidence for such components, increasing concentrations of WT1 repressor domain without a zinc finger DNA binding domain were co-transfected with fixed concentrations of wild-type (wt) WT1 and PDGF A-chain promoter/reporter gene constructs. As levels of the repressor domain were increased, a progressive loss of wt WT1 repressor activity and a progressive increase in its activation were observed, suggesting that the repressor domain of WT1 competes with wt WT1 for an interactive protein that is an essential component of the repressor activity of wt WT1. Because the most common mutation associated with Denys-Drash Syndrome disrupts the zinc finger domains of WT1, the results also suggest that the mutant WT1 may have aberrant DNA binding activity and perhaps function as a dominant negative effector of wt WT1.

Products of alternatively spliced transcripts of the Wilms' tumor suppressor gene, wt1, have altered DNA binding specificity and regulate transcription in different ways.

The Wilms' tumor susceptibility gene, wt1, encodes a transcription factor of the zinc finger protein family. Mutations in the WT1 gene product have been detected in both sporadic and familial Wilms' tumors, suggesting that alterations in WT1 may disrupt its normal function as a transcriptional regulator. The transcripts of wt1 are alternatively spliced; however, roles of the alternatively spliced forms have not been defined. The major transcript of wt1 encodes a WT1 protein [WT1(+KTS)+17AA] that contains three amino acids (+KTS) between the third and fourth zinc fingers and a serine-rich, 17 amino acid (+17AA) domain N-terminal to the zinc finger region. We now show that the WT1 (+KTS) forms functionally bind to a unique G+C-rich sequence within the PDGF A-chain promoter. We also show that WT1 (+KTS)+17AA functions as a strong transcriptional repressor and that +17AA alone fused to the zinc-finger domain of WT1 or to the heterologous DNA binding domain of GAL4 functions independently as a repressor. Deletion of four serine residues within +17AA abolishes the repressor activity of +17AA. These results indicate that wt1 products with +17AA contain an additional dominant repressor domain and that the presence or absence of +KTS determines alternative DNA binding specificity.

Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor.

Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.

[Thrombin-fibrinogen interaction: release of fibrinopeptides and the effects of ATP]. / Interazione trombina-fibrinogeno: rilascio dei fibrinopeptidi ed effetto dell'ATP.

This study presents experimental data on the time dependence of the release of fibrinopeptides A and B following the interaction between thrombin and fibrinogen. It is found that the release of fibrinopeptide A is fast as compared to that of fibrinopeptide B. This process is modulated by ATP in a complex fashion. In fact, the release of fibrinopeptide A is first enhanced at low (less than 1 mM) ATP concentrations and then progressively inhibited at ATP concentrations greater than 1 mM. These results draw attention to a possible model for the in vivo modulation of thrombin activity by ATP.