Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.     

Genetics of Peutz-Jeghers syndrome, Carney complex and other familial lentiginoses

Peutz-Jeghers syndrome (PJS, #175200) and Carney complex (CNC, OMIM#160980) are the two most common multiple neoplasia syndromes associated with lentiginosis. Both disorders are inherited in an autosomal dominant manner and they have recently been elucidated at the molecular level. PJS and CNC share manifestations with Cowden syndrome (or Cowden disease) (CS, OMIM#158350) and Bannayan-Riley-Ruvalcaba syndrome (BRR, OMIM#153480). The endocrine tumors of CS and PJS, which could classify these disorders as variant types of multiple endocrine neoplasias (MENs), are not present in most CS and BRR patients, but lentigines are shared by PJS, CNC and BRR. The serine-threonine kinase STK11 (or LKB1), located on 19p13, is mutated in more than half of all PJS kindreds. The R1alpha subunit of c-AMP-dependent protein kinase A, located on 17q22-24, is mutated in 40% of CNC kindreds. The protein phosphatase PTEN is mutated in most cases of CS and in almost 50% of BRR kindreds, despite significant clinical heterogeneity in these syndromes. The molecular elucidation of the lentiginoses and their related syndromes identifies new pathways of growth control and cellular regulation that are important for endocrine signaling, tumorigenesis, cutaneous function and embryonic development.     

Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. There is an increased risk of benign and malignant tumors in the gastrointestinal tract and in extraintestinal tissues. One PJS locus has been mapped to chromosome 19p13.3; a second locus is suspected on chromosome 19q13.4 in a minority of families. The PJS gene on 19p13.3 has recently been cloned, and it encodes the serine/threonine kinase LKB1. The gene, which is ubiquitously expressed, is composed of 10 exons spanning 23 kb. Several LKB1 mutations have been reported in heterozygosity in PJS patients. In this study, we screened for LKB1 mutations in nine PJS families of American, Spanish, Portuguese, French, Turkish, and Indian origin and detected seven novel mutations. These included two frameshift mutations, one four-amino-acid deletion, two amino-acid substitutions, and two splicing errors. Expression of mutant LKB1 proteins (K78I, D176N, W308C, and L67P) and assessment of their autophosphorylation activity revealed a loss of the kinase activity in all of these mutants. These results provide direct evidence that the elimination of the kinase activity of LKB1 is probably responsible for the development of the PJS phenotypes. In two Indian families, we failed to detect any LKB1 mutation; in one of these families, we previously had detected linkage to markers on 19q13.3-4, which suggests locus heterogeneity of PJS. The elucidation of the molecular etiology of PJS and the positional cloning of the second potential PJS gene will further elucidate the involvement of kinases/phosphatases in the development of cancer-predisposing syndromes.     

宫颈癌患者高危型人乳头瘤病毒感染 与免疫功能和癌基因表达的相关性

摘要：目的 探讨宫颈癌患者高危型人乳头瘤病毒（HPV）感染的检测及其与免疫功能和癌基因表达的相关性。方法 选择2017年9月至2018年12月在我院接受手术治疗的118例原发性宫颈癌患者为宫颈癌组,根据高危型HPV感染情况进一步分为高危型HPV组（89例）和非高危型HPV组（29例）。选择同期在我院行子宫全切术的57例子宫肌瘤患者为子宫肌瘤组。对比各组患者外周血T淋巴细胞亚群分布情况,病灶组织中原癌基因（E6/E7、c-Met、SALL4、PGRN）和抑癌基因（p53、pRb、PTEN、LKB1）表达量的差异。结果 宫颈癌组患者病灶组织中高危型HPV感染率显著高于子宫肌瘤组（75.42% vs 22.81%,2=43.764,P＜0.05）。高危型HPV组患者外周血中CD4+ T淋巴细胞比例、CD4+T/CD8+ T比值低于非高危型HPV组,CD8+ T淋巴细胞比例高于非高危型HPV组（均P＜0.05）。高危型HPV组患者病灶组织中E6/E7、c-Met、SALL4、PGRN mRNA的表达量高于非高危型HPV组,而p53、pRb、PTEN、LKB1 mRNA的表达量低于非高危型HPV组（均P＜0.05）。结论 高危型HPV感染可导致宫颈癌患者细胞免疫功能下降及肿瘤恶性程度增加,可能是导致病情恶化、治疗效果不佳的危险因素之一。     

Correlation of staining for LKB1 and COX-2 in hamartomatous polyps and carcinomas from patients with Peutz-Jeghers syndrome.

Germline mutations of the LKB1 gene lead to Peutz-Jeghers syndrome (PJS), which is associated with a predisposition to gastrointestinal polyposis and cancer. In this study we tested for germline mutations of LKB1 in 11 patients with PJS from nine families and analyzed the expression patterns of the LKB1 and cyclo-oxygenase-2 (COX-2) proteins in 28 Peutz-Jeghers polyps (PJPs) and five carcinomas from these patients by immunohistochemical (IHC) analysis. In eight of those families we identified seven different mutations, which consisted of two splice site mutations, two nonsense mutations, one small in-frame deletion, one frame-shift mutation, and one silent mutation. Immunostaining revealed nuclear and cytoplasmic expression of LKB1 protein in 23 PJPs and five carcinomas, nuclear expression alone in one PJP, and loss of LKB1 protein expression in four PJPs, indicating a heterogeneous LKB1 expression pattern in PJPs. Overexpression of COX-2 was detected in 23 (82%) of 28 PJPs and in all carcinomas. Despite heterogeneity in staining of LKB1 among individuals and even among samples from the same individual, we found statistically significant correlations in staining of LKB1 relative to COX-2. These results suggest that COX-2 plays a role in tumorigenesis in PJS and may therefore be considered as a potential target for PJS chemoprevention.     

LKB1 Catalytic Activity Contributes to Estrogen Receptor α Signaling

The tumor suppressor serine-threonine kinase LKB1 is mutated in Peutz-Jeghers syndrome (PJS) and in epithelial cancers, including hormone-sensitive organs such as breast, ovaries, testes, and prostate. Clinical studies in breast cancer patients show low LKB1 expression is related to poor prognosis, whereas in PJS, the risk of breast cancer is similar to the risk from germline mutations in breast cancer (BRCA) 1/BRCA2. In this study, we investigate the role of LKB1 in estrogen receptor α (ERα) signaling. We demonstrate for the first time that LKB1 binds to ERα in the cell nucleus in which it is recruited to the promoter of ERα-responsive genes. Furthermore, LKB1 catalytic activity enhances ERα transactivation compared with LKB1 catalytically deficient mutants. The significance of our discovery is that we demonstrate for the first time a novel functional link between LKB1 and ERα. Our discovery places LKB1 in a coactivator role for ERα signaling, broadening the scientific scope of this tumor suppressor kinase and laying the groundwork for the use of LKB1 as a target for the development of new therapies against breast cancer.     

Developmentally regulated expression of the mouse homologues of the potassium channel encoding genes m-erg1, m-erg2 and m-erg3

Deciphering the expression pattern of K+ channel encoding genes during development can help in the understanding of the establishment of cellular excitability and unravel the molecular mechanisms of neuromuscular diseases. We focused our attention on genes belonging to the erg family, which is deeply involved in the control of neuromuscular excitability in Drosophila flies and possibly other organisms. Both in situ hybridisation and RNase Protection Assay experiments were used to study the expression pattern of mouse (m)erg1, m-erg2 and m-erg3 genes during mouse embryo development, to allow the pattern to be compared with their expression in the adult. M-erg1 is first expressed in the heart and in the central nervous system (CNS) of embryonic day 9.5 (E9.5) embryos; the gene appears in ganglia of the peripheral nervous system (PNS) (dorsal root (DRG) and sympathetic (SCG) ganglia, mioenteric plexus), in the neural layer of retina, skeletal muscles, gonads and gut at E13.5. In the adult m-erg1 is expressed in the heart, various structures of the CNS, DRG and retina. M-erg2 is first expressed at E9.5 in the CNS, thereafter (E13.5) in the neural layer of retina, DRG, SCG, and in the atrium. In the adult the gene is present in some restricted areas of the CNS, retina and DRG. M-erg3 displayed an expression pattern partially overlapping that of m-erg1, with a transitory expression in the developing heart as well. A detailed study of the mouse adult brain showed a peculiar expression pattern of the three genes, sometimes overlapping in different encephalic areas.     

BCS1L is expressed in critical regions for neural development during ontogenesis in mice

BCS1L is a chaperone necessary for the incorporation of Rieske FeS and Qcr10p into complex III (CIII) of the respiratory chain. Mutations in the BCS1L gene cause early fetal growth restriction and a lethal neonatal disease in humans, however, the pathogenesis remains unclear. Here, we analysed the expression of BCS1L during mouse embryonic development and compared its expression with that of the mitochondrial markers Porin, GRIM19, Core I, and Rieske FeS. BCS1L was strongly expressed in embryonic tissues already at embryonic days 7 (E7) and 9 whereas the expression of Porin and Rieske FeS was not as evident at this time point. At E11, BCS1L, Porin, and Rieske FeS had overlapping expression patterns in organs known to contain high numbers of mitochondria such as heart, liver and somites. In contrast, BCS1L was differently distributed compared to the mitochondrial proteins Porin, Rieske FeS, Core I and Grim 19 in the floor plate of the E11, E12 and E13 neural tube. These results show that the expression pattern of BCS1L only partially overlaps with the expression of Porin and Rieske FeS. Thus, BCS1L alone or in cooperation with Rieske FES may during development have previously unknown functions beside its role in assembly of complex III. The floor plate of the neural tube is essential for dorsal ventral patterning and the guidance of the developing neurons to their targets. The predominant expression of BCS1L in this region, together with its presence in peripheral ganglia from E13 onwards, indicates a role for BCS1L in the development of neural structures.     

The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death.

Here, we investigate the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). We demonstrate that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. We propose that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients.     

An LKB1-interacting protein negatively regulates TNFα-induced NF-κB activation

The Peutz-Jeghers syndrome (PJS) is a hereditary disorder that predisposes an individual to benign and malignant tumors in multiple organ systems. Recently, the locus responsible for PJS was mapped genetically to the LKB1 gene, with a subsequent investigation proving that it is responsible for most cases of PJS. LKB1 encodes a nuclear serine/threonine protein kinase, and potential tumor-suppressing activity has been attributed to LKB1 kinase. However, how LKB1 exerts its tumor-suppressing function remains to be determined. In this report, we describe the identification of a putative human LKB1-interacting protein, FLIP1, using the yeast two-hybrid system. Two regions of the LKB1 sequence have been determined to be crucial for the interaction with FLIP1. FLIP1 encodes a protein of 429 amino acids with a predicted molecular weight of 47 kd. In contrast to LKB1, which is mainly nuclear, FLIP1 is a cytoplasmic protein, and its expression is ubiquitous in all human tissues examined to date. Interestingly, deletion of the 195 N- terminal amino acids allows FLIP1 to enter the nucleus, suggesting the presence of a regulatory mechanism through its N-terminus for nuclear entry. In addition, we found that ectopic expression of FLIP1 selectively blocks cytokine-induced NF-kappaB activation. The involvement of FLIP1 in the regulation of NF-kappaB activity may shed new light on the role of LKB1 in tumor suppression.     

Search for the second Peutz-Jeghers syndrome locus: exclusion of the STK13, PRKCG,KLK10, and PSCD2 genes on chromosome 19 and the STK11IP gene on chromosome 2

Pathogenic mutations in the serine/threonine kinase STK11 (alias LKB1) cause Peutz-Jeghers syndrome (PJS) in most affected individuals. However, in a considerable number of PJS-patients mutations cannot be detected in STK11 suggesting genetic heterogeneity. One PJS family without STK11 mutations (PJS07) has previously been described with significant evidence for linkage to a second potential PJS locus on 19q13.3-->q13.4. In this study we investigated candidate genes within markers D19S180 and D19S254, since multipoint linkage analysis yielded significant LOD scores for this region in this family. Four genes in the region (cytohesin 2: PSCD2, kallikrein 10: KLK10, protein kinase C gamma: PRKCG, and serine/threonine kinase 13: STK13) potentially involved in growth inhibitory pathways or in the pathophysiology of can- cer, were considered as candidates. We first determined the genomic structure of the PSCD2 and PRKCG genes, and performed mutation analysis of all exons and exon-intron junctions of the four genes, in the PJS07 family. No pathogenic mutation was identified in these four genes in affected individuals. A very rare polymorphism resulting in a conserved amino acid change Lys to Arg was found in PSCD2. These data provide considerable evidence for exclusion of these four genes as candidates for the second locus on 19q13.3-->q13.4 in PJS. Finally, we also excluded the recently identified STK11-interacting protein gene (STK11IP, alias LIP1) mapped in 2q36 as candidate for PJS in the PJS07 family, although this could be a good candidate in other non-STK11/LKB1 families.     

Distinct expression of midkine and pleiotrophin in the spinal cord and placental tissues during early mouse development

Midkine and pleiotrophin comprise a family of heparin-binding growth factors, and are expressed in overlapping tissues during the mid- to late-gestation periods of mouse development. Their distinct expression during early mouse development, as revealed by in situ hybridization, was reported. Midkine was expressed in the embryonic ectoderm from as early as embryonic day (E5.5). In the neural tube midkine was expressed specifically in the neuroepithelium, that is, in the whole area of the neural tube at E9.5, and in the ventricular zone from E10.5-13.5. At E15.5, when the neuroepithelium disappeared, midkine concomitantly became undetectable. In contrast, pleiotrophin expression started exclusively in the neural plate at E8.5, and in the lateral plate of the neural tube at E9.5. It then became restricted to a dorsal ventricular zone from E11.5-13.5, and finally to the central gray neurons at E15.5. Moreover, pleiotrophin was expressed in the ventral horns. Among placental tissues, midkine was detected in the chorion, the fetal component of the placenta, whereas pleiotrophin was found in the decidua basalis, the maternal component of the placenta. The distinct expression of midkine and pleiotrophin suggests their differential role in early development.     

LKB1 (XEEK1) regulates Wnt signalling in vertebrate development

Germline LKB1/STK11 mutations are associated with the cancer-prone Peutz-Jeghers syndrome (PJS) in humans, and nullizygosity provokes a poorly understood constellation of developmental perturbations in the mid-gestational mouse. To gain a better understanding of the processes regulated by LKB1, we have exploited the experimental merits of the developing Xenopus embryo. Here, specific inhibition of XEEK1, the Xenopus orthologue of LKB1, engendered developmental anomalies - shortened body axis and defective dorsoanterior patterning - associated previously with aberrant Wnt signalling. In line with this, LKB1/XEEK1 cooperates with the Wnt-beta-catenin signalling in axis induction and modulates the expression of Wnt-responsive genes in both Xenopus embryos and mammalian cells. We establish that LKB1/XEEK1 acts upstream of beta-catenin in the Wnt-beta-catenin pathway in vivo. LKB1/XEEK1 regulates glycogen synthase kinase (GSK)3beta phosphorylation and it is physically associated in vivo with GSK3beta and protein kinase C (PKC)-zeta, a known GSK3 kinase. These studies show that LKB1/XEEK1 is required for Wnt-beta-catenin signalling in frogs and mammals and provides novel insights into its role in vertebrate developmental patterning and carcinogenesis.     

Loss of cytoplasmic retention ability of mutant LKB1 found in Peutz-Jeghers syndrome patients.

LKB1 Serine/Threonine (ST) kinase (also called STK11) originally identified in our novel protein kinase search project has recently been recognized as a susceptibility gene of Peutz-Jeghers Syndrome (PJS; MIM 175200). PJS is a dominantly inherited human disorder which is characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. Since PJS patients also show a predisposition to a wide spectrum of cancers, it is speculated that LKB1 has a tumor suppressor function. In the present study we have characterized the basic biochemical property of LKB1. In the analysis of mutant LKB1 identified in PJS patients, it was found that one of the mutants, SL26, does not lose its kinase function, but alters its subcellular distribution to accumulate in the nucleus only, whereas wild type LKB1 shows both nuclear and cytoplasmic localization. Domain mapping of the nuclear targeting signal of LKB1 assigned it to its amino terminal side. Furthermore, it was shown that LKB1 also has a cytoplasmic retention ability which is considered defective and pathogenic in the SL26 mutant. It is speculated that subcellular distribution of LKB1 is regulated in the balance of these two forces, importation into the nucleus and retention within the cytoplasm; and the cytoplasmic retention ability is necessary for LKB1 to fulfil its normal function.     

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.     

Liver kinase B1 (LKB1) in the pathogenesis of UVB-induced murine basal cell carcinoma

LKB1, a known tumor suppressor, is mutated in Peutz–Jeghers Syndrome (PJS). It is responsible for the enhanced cancer risk in patients with PJS. Dysregulation of LKB1-dependent signaling also occurs in various epithelial cancers. UVB alters the expression of LKB1, though its role in the pathogenesis of skin cancer is unknown. Here we describe upregulation of LKB1 expression in UVB-induced murine basal cell carcinoma (BCC) and in human skin tumor keratinocytes. AMP-kinase and acetyl Co-A carboxylase, the downstream LKB1 targets, are also enhanced in this neoplasm. In addition, p-Akt, a kinase which inactivates GSK3β by its phosphorylation, is enhanced in BCCs. Consistently, an accumulation of p-GSK3β and an increase in activated nuclear β-catenin are found. mTOR signaling, which is also inhibited by LKB1, remains upregulated in BCCs. However, a marked decrease in the expression of sestrins, which function as potent negative regulators of mTOR is observed. Metformin, a known chemical inducer of this pathway, was found effective in immortalized HaCaT keratinocytes, but failed to activate the LKB1-dependent signaling in human carcinoma A431 cells. Thus, our data show that the LKB1/AMPK axis fails to regulate mTOR pathway, and a complex regulatory mechanism exists for the persistent mTOR activation in murine BCCs.     

The Drosophila innexin 7 gap junction protein is required for development of the embryonic nervous system

The Drosophila genome encodes eight members of the innexin family of gap junction proteins. Most of the family members are expressed in complex and overlapping expression patterns during Drosophila development. Functional studies and mutant analysis have been performed for only few of the innexin genes. The authors generated an antibody against Innexin7 and studied its expression and functional role in embryonic development by using transgenic RNA interference (RNAi) lines. The authors found Innexin7 protein expression in all embryonic epithelia from early to late stages of development, including in the developing epidermis and the gastrointestinal tract. In early embryonic stages, the authors observed a nuclear localization of Innexin7, whereas Innexin7 was found in a punctuate pattern in the cytoplasm and at the membrane of most epithelial tissues at later stages of development. During central nervous system (CNS) development, Innexin7 was expressed in cells of the neuroectoderm and the mesectoderm and at later stages of embryogenesis, its expression was largely restricted to a segmental pattern of few glia and neuronal cells derived from the midline precursors. Coimmunostaining experiments showed that Innexin7 is expressed in midline glia, and in two different neuronal cells, the pCC and MP2 neurons, which are pioneer cells for axon guidance. RNAi-mediated knock down was used to gain insight into the embryonic function of innexin7. Down-regulation of innexin7 expression resulted in a severe disruption of embryonic nervous system development. Longitudinal, posterior, and anterior commissures were disrupted and the outgrowth of axon fibers of the ventral nerve cord was aberrant, causing peripheral nervous system defects. The results suggest an essential role for innexin7 for axon guidance and embryonic nervous system development in Drosophila.