Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane.

Basement membranes are important for epithelial differentiation, cell survival, and normal and metastatic cell migration. Much is known about their breakdown and remodeling, yet their positive regulation is poorly understood. Our previous analysis of a fibroblast growth factor (FGF) receptor mutation raised the possibility that protein kinase B (Akt/PKB) activated by FGF is connected to the expression of certain laminin and type IV collagen isotypes. Here we test this hypothesis and demonstrate that constitutively active Akt/PKB, an important downstream element of phosphoinositide 3'-kinase signaling, induces the synthesis of laminin-1 and collagen IV isotypes and causes their translocation to the basement membrane. By using promoter-reporter constructs, we show that constitutively active phosphoinositide 3'-kinase-p110 or Akt/PKB activates, whereas dominant negative Akt/PKB inhibits, transcription of laminin beta1 and collagen IV alpha1 in differentiating C2 myoblast- and insulin-induced Chinese hamster ovary-T cell cultures. These results suggest that Akt/PKB activated by receptor tyrosine kinases is involved in the positive regulation of basement membrane formation. The possible role of Akt/PKB-induced laminin and collagen IV synthesis in cell survival and differentiation will be discussed.

Modulation of extracellular matrix adhesiveness by neurocan and identification of its molecular basis.

Neurocan is one of the major chondroitin sulfate proteoglycans of perinatal rodent brain. HEK-293 cells producing neurocan recombinantly show changes in their behavior. The expression of full-length neurocan led to a detachment of the secreting cells and the formation of floating spheroids. This occurred in the continuous presence of 10% fetal bovine serum in the culture medium. Cells secreting fragments of neurocan-containing chondroitin sulfate chains and the C-terminal domain of the molecule showed a similar behavior, whereas cells expressing fragments of neurocan-containing chondroitin sulfate chains but lacking parts of the C-terminal domain did not show spheroid formation. Cells secreting the hyaluronan-binding N-terminal domain of neurocan showed an enhanced adhesiveness. When untransfected HEK-293 cells were plated on a surface conditioned by spheroid-forming cells, they also formed spheroids. This effect could be abolished by chondroitinase treatment of the conditioned surface. The observations indicate that the ability of the chondroitin sulfate proteoglycan neurocan to modulate the adhesive character of extracellular matrices is dependent on the structural integrity of the C-terminal domain of the core protein.

Neonatal exposure to neurotoxic pesticides increases adult susceptibility: a review of current findings.

An environmental mischance commonly occuring in nature is the combination of neonatal exposure and later adult exposure to various toxic substances. During neonatal life, offspring can be affected by toxic agents either by transfer via mother's milk or by direct exposure. In many mammalian species the perinatal period is characterized by a rapid development of the brain--'the brain growth spurt' (BGS). We have observed that exposure to pesticides, such as DDT and bioallethrin, during the BGS in mice can potentiate susceptibility to bioallethrin or paraoxon in adult life. This combined neonatal and adult exposure caused spontaneous behavioural aberrations and changes in muscarinic cholinergic receptors and led to impairment of the faculties of learning and memory. Our studies indicate that neonatal exposure to pesticides--even in low doses--can potentiate and/or modify the reaction to adult exposure to xenobiotics, and thereby accelerate dysfunctional processes.

Differential expression of muscarinic subtype mRNAs after exposure to neurotoxic pesticides.

We have recently reported an increase in the density of muscarinic cholinergic receptors in mice neonatally exposed to a persistent environmental agent, dichlorodiphenyltrichloroethane (DDT), and a subsequent exposure as adults to nonpersistent toxicants, such as bioallethrin or paraoxon. Here we have examined the effects of an exposure like this on muscarinic receptor mRNA expression. Ten-day-old Naval Medical Research Institute mice received a single oral dose of DDT (0.5 mg/kg body weight). When aged 5 months, they received bioallethrin (0.7 mg/kg body weight per day for 7 days) or paraoxon (1.4 mg/kg body weight every second day for 7 days). mRNA expression of subtypes m1, m3, and m4 was studied in 7-month-old animals. Changes could only be discovered in the DDT-bioallethrin treated mice, where expression of subtype m4 was elevated in cortex and caudate putamen. Moreover, the expression pattern of the subtypes m1, m3, and m4 in mouse brains was found to be very similar to that seen in rats, except for slight differences in the pyramidal cell layer of the hippocampus, where the outermost part of the CA3 region did not show any m4 hybridization. The present study indicates that the earlier observed increase in muscarinic receptor density in mice exposed as neonates to DDT and as adults to bioallethrin can be attributed to changes in the expression of m4.

Changes in behavior and muscarinic receptor density after neonatal and adult exposure to bioallethrin.

Throughout life, mammals are exposed to environmental toxicants, some of which have acute effects on the nervous system. Early, low-dose exposure in combination with later re-exposure and possible interference with normal aging have been little studied. The present study revealed increased susceptibility in adult mice, exposed neonatally to a low dose of the insecticide bioallethrin, to renewed exposure to bioallethrin as adults. Ten-day-old Naval Medical Research Institute male mice received bioallethrin orally (0.7 mg per kg body weight per day for 7 days). When aged 5 months they were given the same dose of bioallethrin by gavage. Twenty-four hours after the last administration, a spontaneous motor activity test revealed significant aberrations in mice exposed both neonatally and as adults to bioallethrin. The density of muscarinic receptors was significantly increased. When aged 7 months, spontaneous behavioral disturbances and muscarinic receptor changes persisted and learning and memory deficits had developed. These results indicate that neonatal exposure to bioallethrin has the potential to increase susceptibility of the adult mouse to a new exposure at a dosage that does not have any effect in animals treated neonatally with vehicle.