Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice.

The LKB1 tumor suppressor gene, mutated in Peutz-Jeghers syndrome, encodes a serine/threonine kinase of unknown function. Here we show that mice with a targeted disruption of Lkb1 die at midgestation, with the embryos showing neural tube defects, mesenchymal cell death, and vascular abnormalities. Extraembryonic development was also severely affected; the mutant placentas exhibited defective labyrinth layer development and the fetal vessels failed to invade the placenta. These phenotypes were associated with tissue-specific deregulation of vascular endothelial growth factor (VEGF) expression, including a marked increase in the amount of VEGF messenger RNA. Moreover, VEGF production in cultured Lkb1(-/-) fibroblasts was elevated in both normoxic and hypoxic conditions. These findings place Lkb1 in the VEGF signaling pathway and suggest that the vascular defects accompanying Lkb1 loss are mediated at least in part by VEGF.

Developmentally regulated expression of Smad3, Smad4, Smad6, and Smad7 involved in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) signaling is mediated from serine/threonine kinase receptors to transcriptional responses via Smad proteins. Here comparison of mRNA expression of Smad3-7 in mouse embryos (E9-E15) revealed developmentally regulated distinct expression patterns for Smad3, 4, 6, and 7. Smad3 was prominently expressed in the differentiating (from E10) central nervous system, but also in developing bones, branchial arches and epithelium of various tissues. Smad4 mostly showed ubiquitous expression, but in E15 embryos, a pronounced signal appeared in epithelial crypts of the gut. Inhibitory Smad6 and Smad7 were coexpressed at high levels in developing cardiovascular system from the earliest stages studied. In contrast, Smad6 was selectively expressed at high levels, e.g. in intramembranous bone whereas Smad7 was prominent in seminiferous tubules of the testis, demonstrating distinct expression of these genes in non-cardiovascular tissues.

Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest.

Germ-line mutations of LKB1 (STK11) lead to Peutz-Jeghers syndrome characterized by gastrointestinal polyps and cancer of different organ systems. The mutations lead to loss or severe impairment of Lkb1 serine/threonine kinase activity. Therefore LKB1 has been implicated as a tumor suppressor gene, but only a few mutations in the coding exons of LKB1 have been detected in sporadic tumors. Here, we have identified tumor cell lines with severely reduced mRNA levels and impaired Lkb1 kinase activity. Reintroducing Lkb1 into these cells suppressed cell growth. The Lkb1-mediated growth inhibition was caused by a G(1) cell cycle block and was not detected with several naturally occurring Lkb1 mutants. These results indicate that LKB1 has functional and specific growth-suppressing activity.

Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development.

Germ-line mutations of LKB1 and PTEN tumor suppressor genes underlie the phenotypically related Peutz-Jeghers syndrome (PJS) and Cowden disease (CD), respectively. To analyze possible developmental roles of PTEN and LKB1, we have studied their mRNA expression during mouse embryonic development (E7-17.5) by in situ hybridization. Ubiquitous expression of both genes during early stages (E7-11) became more restricted in later embryonic development (E15-19) where LKB1 and PTEN showed prominent overlapping expression in e.g. gastrointestinal tract and lung. In contrast, LKB1 was selectively expressed at high levels in testis and PTEN was prominently expressed in skin epithelium and underlying mesenchyme. These results indicate that LKB1 and PTEN display largely overlapping expression patterns during embryonic development. Moreover, a high expression of these genes was observed in the tissues and organs affected in PJS and CD patients and in PTEN+/- mice.

Mutations and impaired function of LKB1 in familial and non-familial Peutz-Jeghers syndrome and a sporadic testicular cancer.

Germline mutations in LKB1 have been reported to underlie familial Peutz-Jeghers syndrome (PJS) with intestinal hamartomatous polyps and an elevated risk of various neoplasms. To investigate the prevalence of LKB1 germline mutations in PJS more generally, we studied samples from 33 unrelated PJS patients including eight non-familial sporadic patients, 20 familial patients and five patients with unknown family history. Nineteen germline mutations were identified, 12 (60%) in familial and four (50%) in sporadic cases. LKB1 mutations were not detected in 14 (42%) patients, indicating that the existence of additional minor PJS loci cannot be excluded. LKB1 is predicted to encode a serine/threonine kinase. To demonstrate the putative Lkb1 kinase function and to study the consequences of LKB1 mutations in PJS and sporadic tumors, we have analyzed the kinase activity of wild-type and mutant Lkb1 proteins. Interestingly, while most of the small deletions or missense mutations resulted in loss-of-function alleles, one missense mutation (G163D) previously identified in a sporadic testicular tumor demonstrated severely impaired but detectable kinase activity.

Central inhibition of nitric oxide synthesis increases blood pressure and heart rate in anesthetized rats.

In the present study we evaluated the cardiovascular responses to inhibition of endogenous nitric oxide (NO) formation in the brain with intracerebroventricular (i.c.v) administration of N omega-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of NO synthase. L-NAME (30 micrograms and 300 micrograms i.c.v) induced a dose-dependent increase in mean arterial pressure and heart rate in anesthetized normotensive rats, while its enantiomer D-NAME (300 micrograms i.c.v.) increased blood pressure only slightly and transiently. The pressor response to L-NAME was partially attenuated by i.c.v. administration of NO precursor L-arginine (300 micrograms), whereas D-arginine, the stereoisomer which cannot serve as a percursor for the biosynthesis of NO, was ineffective. Inhibition of beta 1-adrenoceptors by pretreatment with atenolol (2.5 mg/kg i.v.) reduced the pressor and tachycardic effect of subsequently administered L-NAME, whereas muscarinic receptor antagonist methylatropine (2 mg/kg i.v.) did not affect the cardiovascular effects of L-NAME. These findings imply that the pressor response to i.c.v. L-NAME results from withdrawal of the inhibitory effect of endogenous NO on a central pressor mechanism which acts by increasing sympathetic outflow.

Arg506Gln factor V mutation (factor V Leiden) in patients with ischaemic cerebrovascular disease and survivors of myocardial infarction.

The point mutation Arg506- > Gln of factor V was recently shown to be an important and relatively common genetic cause of venous thromboembolism. Using a DNA technique based on polymerase chain reaction, we surveyed the blood samples of 236 patients with ischaemic stroke or a transient ischaemic attack, 122 survivors of myocardial infarction and 137 control subjects for the presence of this mutation. Although the frequency of the factor V mutation in patients with arterial disease (4.5%) was not significantly different from that in healthy blood donors (2.9%), a carrier status for this mutant gene was associated with symptoms of migraine and relatively mild angiographic abnormalities among patients with cerebrovascular disease. A more extensive study addressing the occurrence and significance of the mutant factor V mutation in patients with vasospastic cerebrovascular diseases seems to be warranted.