A new perspective on the immune escape mechanism in HCC: onco-foetal reprogramming.

We found a shared immunosuppressive microenvironment between foetal liver and hepatocellular carcinoma (HCC) which includes the re-emergence of foetal-associated endothelial cells (PLVAP/VEGFR2) and foetal-like (FOLR2) tumour-associated macrophages in HCC, mediated via VEGF-NOTCH signalling. The discoveries suggest possible novel targets for therapeutic interventions in HCC.

Multimodality Therapy Including Proton Beam Therapy for AFP Producing Esophageal Cancer with Multiple Liver Metastases.

A 50-year-old man who presented with a fever and epigastralgia was diagnosed to have esophageal carcinoma which was identified as poorly differentiated adenocarcinoma producing alpha-fetoprotein (AFP) with Barrett's esophagus. Computed tomography revealed multiple liver metastases and lymph node metastases surrounding the stomach. We first performed chemotherapy for the systemic lesions and proton beam therapy for the local control of lesions without complete remission and we were able to successfully control the frequently recurring lesions by proton beam therapy, cryotherapy and chemotherapy. A complete response has been maintained for 16 months and the overall survival time is 4 years and 2 months. Proton beam therapy for primary esophageal cancer and metastatic lesions was thus found to be an effective therapeutic option for such cases.

Structural basis for alpha fetoprotein-mediated inhibition of caspase-3 activity in hepatocellular carcinoma cells.

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients.

Alpha fetoprotein antagonizes apoptosis induced by paclitaxel in hepatoma cells in vitro.

Hepatocellular carcinoma (HCC) cell resistance to the effects of paclitaxel has not been adequately addressed. In this study, we found that paclitaxel significantly inhibited the viability of HLE, Bel 7402 and L-02 cells in a dose- and time-dependent manner. HLE cells and L-02 cells resisted the cytotoxicity of paclitaxel when transfected with pcDNA3.1-afp vectors. However, Bel 7402 cell sensitivity to paclitaxel was increased when transfected with alpha fetoprotein (AFP)-siRNA. Bel 7402 cell resistance to paclitaxel was associated with the expression of the "stemness" markers CD44 and CD133. Paclitaxel significantly inhibited growth and promoted apoptosis in HLE cells and L-02 cells by inducing fragmentation of caspase-3 and inhibiting the expression of Ras and Survivin, but pcDNA3.1-afp vectors prevented these effects. However, paclitaxel could not significantly promote the cleavage of caspase-3 or suppress the expression of Ras and Survivin in Bel 7402 cells. Silenced expression of AFP may be synergistic with paclitaxel to restrain proliferation and induce apoptosis, enhance cleavage of caspase-3, and suppress the expression of Ras and Survivin. Taken together, AFP may be an important molecule acting against paclitaxel-inhibited proliferation and induced apoptosis in HCC cells via repressing the activity of caspase-3 and stimulating the expression of Ras and Survivin. Targeted inhibition of AFP expression after treatment with paclitaxel is an available strategy for the therapy of patients with HCC.

Alpha fetoprotein plays a critical role in promoting metastasis of hepatocellular carcinoma cells.

A high level of serum alpha fetoprotein (AFP) is positively associated with human hepatocellular carcinoma (HCC) carcinogenesis and metastasis; however, the function of AFP in HCC metastasis is unknown. This study has explored the effects of AFP on regulating metastatic and invasive capacity of human HCC cells. Forty-seven clinical patients' liver samples were collected and diagnosed; HCC cells line, Bel 7402 cells (AFP-producing) and liver cancer cell line cells (non-AFP-producing) were selected to analyse the role of AFP in the metastasis of HCC cells. The results indicated that high serum concentration of AFP was positively correlated with HCC intrahepatic, lymph nodes and lung metastasis. Repressed expression of AFP significantly inhibited the capability of migration and invasion of Bel 7402 cells, expression of keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), matrix metalloproteinase 2/9 (MMP2/9) and CXC chemokine receptor 4 (CXCR4) were also down-regulated in Bel 7402 cells; migration and invasion, expression of K19, EpCAM, MMP2/9 and CXCR4 were significantly enhanced when HLE cells were transfected with AFP-expressed vector. The results demonstrated that AFP plays a critical role in promoting metastasis of HCC; AFP promoted HCC cell invasion and metastasis via up-regulating expression of metastasis-related proteins. Thus, AFP may be used as a novel therapeutic target for treating HCC patients.

The adenocarcinoma cell surface mucin receptor for alpha-fetoprotein: is the same receptor present on circulating monocytes and macrophages? A commentary.

The mucin family of proteins is largely expressed on sedentary epithelial cells lining the gastrointestinal, pulmonary, and reproductive tracts and their associated organs and malignant tumors. It is less well-known that mucins are also expressed on circulatory cells of the immune and inflammatory systems, such as monocytes, macrophages, leukemic, and lymphoma cells. The epithelial mucins function in (a) protection and lubrication of mucosal linings, (b) cell adhesion and cell-to-cell contact, (c) cell migration and metastasis, and (d) signal transduction. It would be logical to presume that mucins expressed on circulating mononuclear cells could perform similar functions. Recently, it was proposed that the alpha-fetoprotein (AFP) receptor, known to be present on solid epithelial-derived malignant tumor cells, can be identified as a mucin glycoprotein. Interestingly, it was also reported that AFP binds to a receptor on circulating cells and sedentary tumor cells of lymphoreticular origin, especially monocytes associated with lymphomas and leukemias. The primary objective of the present commentary is to present literature-based evidence that some of the cell surface mucins on sedentary epithelial tumor cells and certain mucins expressed on circulating monocytes/macrophages are identical to the AFP receptor. The secondary objective is to discuss the role of AFP and its derived peptides in the growth suppression of adenocarcinomas and lymphomas using the AFP-mucin receptor concept as a key to the mechanism of tumor growth inhibition.

Effects of AFP-activated PI3K/Akt signaling pathway on cell proliferation of liver cancer.

This study aims to investigate effects of alpha-fetoprotein (AFP)-activated phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway on hepatocellular carcinoma cell proliferation. Active cirrhosis patients after hepatitis B infection (n = 20) and viral hepatitis patients with hepatocellular carcinoma (HCC) (n = 20) were selected as the subjects of the present study. Another 20 healthy subjects were selected as the control group. The serum AFP expression and liver tissue PI3K and Akt gene mRNA expression were detected. The hepatoma cell model HepG2 which had a stable expression of AFP gene was used. Real-time quantitative PCR and Western blot and other methods were used to analyze the intracellular PI3K and Akt protein levels. Compared with control group and cirrhosis group, the serum AFP levels in HCC group significantly increased, and the tissue PI3K and Akt mRNA expression also significantly increased. HepG2 cells were intervened using AFP, in which the PIK and Akt protein expression significantly increased. After intervention by use of AFP monoclonal antibodies or LY294002 inhibitor, the PIK and Akt protein expression in HepG2 cell was significantly decreased (P < 0.05). AFP can promote the proliferation of hepatoma cells via activation of PI3K/Akt signaling pathway.

ZBTB20 is involved in liver regeneration after partial hepatectomy in mouse.

BACKGROUND: A better understanding of the molecular mechanisms in liver regeneration holds promise for exploring the new potential therapy for liver failure. The present study was to investigate the role of zinc finger and BTB domain-containing protein 20 (ZBTB20), a potential factor associated with liver regeneration, in a model of 70% hepatectomy in mice. METHODS: Parameters for liver proliferation such as liver/body ratio and BrdU positivity were obtained via direct measurement and immunohistochemistry. The levels of zinc fingers and homeoboxes 2 (ZHX2), ZBTB20, alpha-fetoprotein (AFP) and glypican 3 (GPC3) transcripts in the regenerating liver tissue of a 70% hepatectomy rodent model were monitored by real-time PCR analysis at different time points. Knockdown of ZBTB20 was performed to characterize its regulatory function. RESULTS: A negatively regulating relationship between ZHX2, ZBTB20 and AFP, GPC3 was revealed from 24 to 72 hours after 70% hepatectomy. ZBTB20 appears to negatively regulate AFP and GPC3 transcription since the knockdown of ZBTB20 promoted the proliferation of hepatocytes and the expression of AFP and GPC3. CONCLUSION: In addition to AFP, GPC3 and ZHX2, ZBTB20 is a new regulator in liver regeneration and the decrease of ZBTB20 expression following 70% hepatectomy promotes AFP and GPC3 expression.

Keratin 19: a key role player in the invasion of human hepatocellular carcinomas.

OBJECTIVE: Keratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive. DESIGN: Clinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays. RESULTS: In the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib. CONCLUSIONS: Giving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

Alpha-fetoprotein contributes to THP-1 cell invasion and chemotaxis via protein kinase and Gi-protein-dependent pathways.

Alpha-fetoprotein (AFP) for long was known as immunomodulator and tumor marker having multifaceted actions on the activity of normal and transformed cells. In present study, we have investigated the involvement of AFP in regulation of THP-1 cell line invasion and underlying mechanisms. Treatment with human recombinant AFP causes up-regulation of MMP9 expression, chemotaxis and calcium mobilization, and increases invasion through Matrigel, with no significant impact on THP-1 cell growth or viability. Using small molecule inhibitors, we have shown that the rhAFP-induced MMP9 expression depends on the activation of ERK1,2, JNK and Akt kinases, with the involvement of NFκB and likely, AP-1 transcription factors. In contrast, inhibition of p38 kinase, but not of JNK, had dramatic suppressive effect on the rhAFP-triggered chemotaxis. In addition, rhAFP-induced MMP9 expression and calcium response were completely blocked by pertussis toxin, indicating that Gi-protein-coupled receptor(s) has a mediatory role in these processes. CCR5 chemokine receptor is the only known Gi-protein binding to AFP. The action of CCR5 inhibitor Maraviroc results in partial suppression of MMP9 up-regulation and calcium response suggesting that CCR5 might be involved in these effects.

Female mice deficient in alpha-fetoprotein show female-typical neural responses to conspecific-derived pheromones.

The neural mechanisms controlling sexual behavior are sexually differentiated by the perinatal actions of sex steroid hormones. We recently observed using female mice deficient in alpha-fetoprotein (AFP-KO) and which lack the protective actions of AFP against maternal estradiol, that exposure to prenatal estradiol completely defeminized the potential to show lordosis behavior in adulthood. Furthermore, AFP-KO females failed to show any male-directed mate preferences following treatment with estradiol and progesterone, indicating a reduced sexual motivation to seek out the male. In the present study, we asked whether neural responses to male- and female-derived odors are also affected in AFP-KO female mice. Therefore, we compared patterns of Fos, the protein product of the immediate early gene, c-fos, commonly used as a marker of neuronal activation, between wild-type (WT) and AFP-KO female mice following exposure to male or estrous female urine. We also tested WT males to confirm the previously observed sex differences in neural responses to male urinary odors. Interestingly, AFP-KO females showed normal, female-like Fos responses, i.e. exposure to urinary odors from male but not estrous female mice induced equivalent levels of Fos protein in the accessory olfactory pathways (e.g. the medial part of the preoptic nucleus, the bed nucleus of the stria terminalis, the amygdala, and the lateral part of the ventromedial hypothalamic nucleus) as well as in the main olfactory pathways (e.g. the piriform cortex and the anterior cortical amygdaloid nucleus), as WT females. By contrast, WT males did not show any significant induction of Fos protein in these brain areas upon exposure to either male or estrous female urinary odors. These results thus suggest that prenatal estradiol is not involved in the sexual differentiation of neural Fos responses to male-derived odors.

Alpha-fetoprotein acts as a novel signal molecule and mediates transcription of Fn14 in human hepatocellular carcinoma.

BACKGROUND & AIMS: The function of cytoplasmic AFP as a regulatory factor in the growth of tumor cells has been well defined. However, its precise mechanism of action and its clinical significance remain to be worked out. METHODS: Specimens from HCC patients were analyzed by using immunohistochemistry, co-immunoprecipitation (CoIP), and chromatin immunoprecipitation (ChIP) assays to evaluate the role of AFP in RAR signaling-mediated carcinogenesis. Quantitative real-time reverse transcription PCR, Western blotting, confocal microscopy, CoIP, GST pull-down, siRNA, gene transfection, and ChIP assays were also used for analysis of cell lines. RESULTS: RAR is able to interact with cytoplasmic AFP and binds to the element of the regulatory region of the Fn14 gene in the neoplastic tissue of HCC patients. An assay of hepatocyte cell lines of differing AFP expression showed that cytoplasmic AFP is able to block ATRA-induced nuclear translocation of RAR and expression of the Fn14 gene. Knockdown of AFP in siRNA-transfected HepG2 and Bel7402 cells led to greater binding of RAR to its response element. The expression of the Fn14 gene was therefore up regulated as reflected by increases in mRNA and protein levels. Conversely, transfection of HLE and L02 cells (AFP negative) with the afp gene resulted in apparent reduction of RAR binding to DNA and Fn14 protein. CONCLUSIONS: Demonstration of the involvement of cytoplasmic AFP in RAR-mediated expression of the Fn14 gene strongly indicates AFP plays a signal molecule-like role in the regulation of hepatocellular carcinoma growth.

Impact of intracellular alpha fetoprotein on retinoic acid receptors-mediated expression of GADD153 in human hepatoma cell lines.

The aim of our study was to gain a better understanding of the molecular mechanism underlying the previously unrecognized role of cytoplasmic alpha fetoprotein (AFP) in retinoic acid receptors (RAR) mediated expression and biological effects of GADD153. Using microarray analysis, the expression of the GADD153 gene showed the greatest fold change among apoptosis/growth related genes in response to ATRA. AFP was able to interact with RAR in HepG2 cells, which was undetectable in HLE cells owing to absence of AFP. ATRA promoted nuclei entrance of RAR, expression of GADD153 and apoptosis, and these changes were reversed after transfection with the afp gene or addition of AGN193109. The level of GADD153 was gradually elevated as the effect of AFP was counteracted by increasing dose or prolonging treatment time with ATRA in HepG2 cells. Knockdown of AFP in siRNA-transfected HepG2 cells or over-expression of AFP in afp gene-transfected HLE cells was synchronously associated with up-regulation or down-regulation, respectively, of GADD153 expression. Both ATRA administration and AFP knockdown were each able to promote greater binding of RAR to its response element with consequent elevation of the proportion of apoptotic cells. Conversely, transfection of HLE cells with pcDNA3.1-afp resulted in apparent reduction of RAR binding to DNA and change of biological effect. These data taken together demonstrate the involvement of AFP in RAR-mediated expression and biological effects of GADD153. These findings provide a novel insight into the mechanism underlying the impact of AFP on the RAR signal network.

[Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters.

Alpha-fetoprotein: a new member of intracellular signal molecules in regulation of the PI3K/AKT signaling in human hepatoma cell lines.

Despite its well-defined role as a serum growth factor during fetal liver development and hepatic oncogenesis, the biological significance of cytoplasmic alpha-fetoprotein (AFP) remains incompletely understood. Here, we provide evidence to illustrate that cytoplasmic AFP may function as a regulator in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in human hepatocellular carcinoma cells. The results demonstrated colocalization and interaction of AFP and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in the cytoplasm of AFP-producing Bel 7402 and HepG2 cells, with an interaction distance of 12.6 ± 2.7 Å as determined with the fluorescence resonance energy transfer technique. Knockdown of AFP mRNA or inhibition of AFP expression by all trans-retinoic acid resulted in enhancement of the PTEN level with a synchronous decrease in phosphorylated AKT. Transfection of the afp gene into HLE cells (originally AFP negative) led to a significant activation of AKT signaling. The inhibition of PI3K signaling by LY 294002 was simultaneously reversed by transfection, accompanied by diminution of all trans-retinoic acid-induced upregulation of PTEN and enhancement of cell growth. In conclusion, these results demonstrate that cytoplasmic AFP is involved in regulation of hepatocellular growth and tumorigenesis.

The "thrifty" gene encoding Ahsg/Fetuin-A meets the insulin receptor: Insights into the mechanism of insulin resistance.

Ahsg (fetuin-A) is a 55-59kDa phosphorylated glycoprotein synthesized in the adult predominantly by hepatocytes, from which it enters the circulation. When dysregulated, this glycoprotein operates to influence the clinical sequelae of insulin resistance-type 2 diabetes and cardiovascular disease. The pathological sequelae likely arise from two separable molecular "faces" of Ahsg-one acting at the level of the insulin receptor and a second face influencing ectopic biomineralization in the intima. A detailed understanding of these two functional faces of Ahsg is not yet clear for lack of structural studies. Ahsg has a physiological role in the biomineralization of bone, which when dysregulated can lead to ectopic calcification of soft tissues in the vasculature. Ahsg has a second physiological function in regulating how insulin signals through its receptor, a transmembrane tyrosine kinase. Dysregulation of this "face" of Ahsg results in morbid sequelae such as impaired glucose disposal and fatty liver. Ahsg binds to tandem fibronectin type 3 (Fn3) domains present in the 194 amino acid residue extracellular portion of the ß-subunit of the insulin receptor, distant from the high-affinity pocket formed by two complementing α-subunits where insulin binds. Only two proteins are known to bind directly to the insulin receptor ectodomain - insulin and Ahsg - the former turns on the receptor's intrinsic tyrosine kinase (TK) activity, and the latter shuts it down. Recent X-ray crystallographic studies of the ectodomain of the insulin receptor now sharpen our understanding of the receptor's extracellular α-subunit and linked ß-subunit. Ahsg genotype and its circulating level have been correlated with body morphometrics (obese versus lean and visceral adiposity) in epidemiological studies enrolling thousands of patients. Epidemiological studies from the clinic reveal high levels of circulating Ahsg in insulin resistance and diabetes. This review endeavors to explain how one protein can mediate diverse pathologies, but specifically addresses its metabolic "face" blunting insulin receptor activity, an action leading to insulin resistance.

Characterisation of bovine epiblast-derived outgrowth colonies.

The aim of the present study was to characterise bovine epiblast-derived outgrowth colonies (OCs) with respect to the embryonic origin of their cellular components. Epiblasts were isolated mechanically from bovine Day 12 embryos. Epiblasts were cultured on feeder layers of SNL cells (neomycin-resistant leukaemia inhibitory factor (LIF)-producing STO cells) in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 15% fetal calf serum, 5% KnockOut Serum Replacement, LIF, basic fibroblast growth factor, non-essential amino acids (NEAA) and nucleosides. Samples were fixed on Days 4, 6 and 8 of culture and processed for immunocytochemistry and transmission electron microscopy. Epiblasts formed OCs consisting of a central core of epiblast-like cells with a basal plate of flattened cells extending outwards from the core. The cells of the core showed nuclear octamer-binding transcription factor 4 (OCT4) staining, indicating an epiblast origin, and some also stained positive for cytoplasmic vimentin. Adjacent cells were linked by tight junctions towards the surface of the colony and rested on a basal lamina. The cells of the basal plate predominantly stained for alpha1-fetoprotein (AFP), indicative of a possible hypoblast origin. Only a few cells scattered within the basal plate exhibited cytokeratin 8 staining, indicating a trophectoderm nature. The intensity of OCT4 and vimentin staining within the core had decreased by Day 8 of culture. In conclusion, OCs derived from bovine Day 12 epiblasts display a central core of OCT4-stained cells of a potential epiblast origin surrounded by a basal plate of mainly AFP-stained cells of a potential hypoblast nature.

The alpha-fetoprotein knock-out mouse model suggests that parental behavior is sexually differentiated under the influence of prenatal estradiol.

In rodent species, sexual differentiation of the brain for many reproductive processes depends largely on estradiol. This was recently confirmed again by using the alpha-fetoprotein knockout (AFP-KO) mouse model, which lacks the protective actions of alpha-fetoprotein against maternal estradiol and as a result represents a good model to determine the contribution of prenatal estradiol to the sexual differentiation of the brain and behavior. Female AFP-KO mice were defeminized and masculinized with regard to their neuroendocrine responses as well as sexual behavior. Since parental behavior is also strongly sexually differentiated in mice, we used the AFP-KO mouse model here to ask whether parental responses are differentiated prenatally under the influence of estradiol. It was found that AFP-KO females showed longer latencies to retrieve pups to the nest and also exhibited lower levels of crouching over the pups in the nest in comparison to WT females. In fact, they resembled males (WT and AFP-KO). Other measures of maternal behavior, for example the incidence of infanticide, tended to be higher in AFP-KO females than in WT females but this increase failed to reach statistical significance. The deficits observed in parental behavior of AFP-KO females could not be explained by any changes in olfactory function, novelty recognition or anxiety. Thus our results suggest that prenatal estradiol defeminizes the parental brain in mice.

alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes.

BACKGROUND AND PURPOSE: The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. EXPERIMENTAL APPROACH: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods. KEY RESULTS: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. CONCLUSIONS AND IMPLICATIONS: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Mechanisms linking osteoporosis with cardiovascular calcification.

Cardiovascular calcium deposition is associated with osteoporosis through various potential mechanisms involving molecular regulatory factors at the nanoscale level that govern skeletal bone and cardiovascular tissues. In this article, several possible mechanisms linking cardiovascular calcification and osteoporosis are discussed, including aging, tissue-specific responses to chronic inflammation, flow-limiting atherosclerosis of skeletal end arteries causing ischemic abnormalities in metabolism, shared endogenous regulatory factors that affect the two tissues in a reciprocal manner, and changes in a cysteine protease inhibitor, fetuin. Any or all of these factors and phenomena may contribute to the association.