Human amniotic epithelial stem cells: Hepatic differentiation and regenerative properties in liver disease treatment.

The placenta and the extraembryonic tissues represent a valuable source of cells for regenerative medicine. In particular, the amniotic membrane possesses cells with stem cells characteristics that have attracted research attention. Human amniotic epithelial cells (hAECs) have unique and desirable features that position them over other stem cells, not only because of the unlimited potential supplied of, the easy access to placental tissues, and the minimal ethical and legal barriers associated, but also due to the embryonic stem cells markers expression and their ability to differentiate into the three germ layers. In addition, they are non-tumorigenic and have immunomodulatory and anti-inflammatory properties. Hepatic failure is one of the major causes of morbidity and mortality worldwide. Organ transplantation is the best way to treat acute and chronic liver failure, but there are several associated obstacles. Stem cells have been highlighted as alternative hepatocytes source because of their potential for hepatogenic differentiation. HAECs, in particular, have some properties that make them suitable for hepatocyte differentiation. In this work, we review the general characteristics of the epithelial stem cells isolated from human amniotic membrane as well as their ability to differentiate to hepatic cells. We also revise their regenerative properties, with the focus on their potential application in the liver disease treatment.

Stem cells and COVID-19: are the human amniotic cells a new hope for therapies against the SARS-CoV-2 virus?

A new coronavirus respiratory disease (COVID-19) caused by the SARS-CoV-2 virus, surprised the entire world, producing social, economic, and health problems. The COVID-19 triggers a lung infection with a multiple proinflammatory cytokine storm in severe patients. Without effective and safe treatments, COVID-19 has killed thousands of people, becoming a pandemic. Stem cells have been suggested as a therapy for lung-related diseases. In particular, mesenchymal stem cells (MSCs) have been successfully tested in some clinical trials in patients with COVID-19. The encouraging results positioned MSCs as a possible cell therapy for COVID-19. The amniotic membrane from the human placenta at term is a valuable stem cell source, including human amniotic epithelial cells (hAECs) and human mesenchymal stromal cells (hAMSCs). Interestingly, amnion cells have immunoregulatory, regenerative, and anti-inflammatory properties. Moreover, hAECs and hAMSCs have been used both in preclinical studies and in clinical trials against respiratory diseases. They have reduced the inflammatory response and restored the pulmonary tissue architecture in lung injury in vivo models. Here, we review the existing data about the stem cells use for COVID-19 treatment, including the ongoing clinical trials. We also consider the non-cellular therapies that are being applied. Finally, we discuss the human amniotic membrane cells use in patients who suffer from immune/inflammatory lung diseases and hypothesize their possible use as a successful treatment against COVID-19.

Aquaporins and placenta.

Water is the major component of cells and tissues. The fetal body consists of about 70-90% water and its fluid balance is dependent on the mother. In fact, abortion, premature birth, amniotic fluid volume abnormality, malformation and fetal growth restrictions might result when the homeostasis of the maternal-fetal fluid exchange is disrupted. Thus, maternal-fetal fluid balance is critical during pregnancy. In this sense, several mechanisms, including aquaporins (AQPs) have been reported to play important roles in maternal-fetal fluid balance. AQPs are small membrane proteins (about 30kDa), present in different organs, that increase the permeability of water, as well as other small uncharged molecules to be transported across the bilayer cell membranes. Several aquaporins are expressed in placenta, and aquaporins play key roles in the placental function. Even though aquaporins have a proven crucial role in water homeostasis, the physiological and pathological importance of aquaporins as glycerol channels is not fully understood. This review focuses on advances in our knowledge of the roles of aquaporins in placental cells, particularly the roles of AQP3 and AQP9 in placental metabolism and points to the pathophysiological importance of glycerol channels in placenta, as well as the signal transduction pathways activated by them. Moreover, the regulation of aquaporins expression by different placental hormones, such as leptin and the mechanisms involved will be discussed.

Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells.

The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.

Proliferation and survival of human amniotic epithelial cells during their hepatic differentiation.

Stem cells derived from placental tissues are an attractive source of cells for regenerative medicine. Amniotic epithelial cells isolated from human amnion (hAECs) have desirable and competitive characteristics that make them stand out between other stem cells. They have the ability to differentiate toward all three germ layers, they are not tumorigenic and they have immunosuppressive properties. Although liver transplantation is the best way to treat acute and chronic hepatic failure patients, there are several obstacles. Recently, stem cells have been spotlighted as alternative source of hepatocytes because of their potential for hepatogenic differentiation. In this work, we aimed to study the proliferation and survival of the hAECs during their hepatic differentiation. We have also analyzed the changes in pluripotency and hepatic markers. We differentiated amniotic cells applying a specific hepatic differentiation (HD) protocol. We determined by qRT-PCR that hAECs express significant levels of SOX-2, OCT-4 and NANOG during at least 15 days in culture and these pluripotent markers diminish during HD. SSEA-4 expression was reduced during HD, measured by immunofluorescence. Morphological characteristics became more similar to hepatic ones in differentiated cells and representative hepatic markers significantly augmented their expression, measured by qRT-PCR and Western blot. Cells achieved a differentiation efficiency of 75%. We observed that HD induced proliferation and promoted survival of hAECs, during 30 days in culture, evaluated by 3H-thymidine incorporation and MTT assay. HD also promoted changes in hAECs cell cycle. Cyclin D1 expression increased, while p21 and p53 levels were reduced. Immunofluorescence analysis showed that Ki-67 expression was upregulated during HD. Finally, ERK 1/2 phosphorylation, which is intimately linked to proliferation and cell survival, augmented during all HD process and the inhibition of this signaling pathway affected not only proliferation but also differentiation. Our results suggest that HD promotes proliferation and survival of hAECs, providing important evidence about the mechanisms governing their hepatic differentiation. We bring new knowledge concerning some of the optimal transplantation conditions for these hepatic like cells.

Leptin upregulates aquaporin 9 expression in human placenta in vitro.

Aquaporins are integral membrane proteins that have permeability functions in many tissues. Aquaporin 9 may transport not only water but also small molecules, such as glycerol, monocarboxylates, purines and pyrimidines. Aquaporin 9 is expressed in syncytiotrophoblast of human term placenta, and it may contribute to the embryonic/fetal growth and survival. We have previously found that Aquaporin 9 expression levels seem to be increased in placenta from gestational diabetes. Since leptin plasma levels and leptin expression are increased in placenta from gestational diabetes, we aimed to study the possible role of leptin on Aquaporin 9 expression in human placenta in vitro. The present work shows that leptin produces a dose-dependent increase of Aquaporin 9 expression, resulting in an increase in Aquaporin-9 protein in human trophoblast explants.

Involvement of leptin in the molecular physiology of the placenta.

Leptin is a homeostatic regulator in the placenta where it promotes proliferation, protein synthesis and the expression of tolerogenic maternal response molecules such as HLA-G. Leptin also exerts an anti-apoptotic action in placenta controlling the expression of p53 master cell cycle regulator under different stress conditions. On the other hand, leptin is an integrative target of different placental stimuli. The expression of leptin in placenta is regulated by hCG, insulin, steroids, hypoxia and many other growth hormones, suggesting that it might have an important endocrine function in the trophoblastic cells. The leptin expression is induced involving the cAMP/PKA or cAMP/Epac pathways which have profound actions upon human trophoblast function. The activation of PI3K and MAPK pathways also participates in the leptin expression. Estrogens play a central role during pregnancy, particularly 17ß-estradiol upregulates the leptin expression in placental cells through genomic and non-genomic actions. The leptin promoter analysis reveals specific elements that are active in placental cells. The transcription factors CREB, AP1, Sp1, NFκB and the coactivator CBP are involved in the placental leptin expression. Moreover, placental leptin promoter is a target of epigenetic marks such as DNA methylation and histone acetylation that regulates not only the leptin expression in placenta during pregnancy but also determines the predisposition of acquiring adult metabolism diseases. Taken together, all these results allow a better understanding of leptin function and regulatory mechanisms of leptin expression in human placental trophoblasts, and support the importance of leptin during pregnancy and in programming adult health.

Leptin reduces apoptosis triggered by high temperature in human placental villous explants: The role of the p53 pathway.

Maternal fever is common during pregnancy and has for many years been suspected to harm the developing fetus. Whether increased maternal temperature produces exaggerated apoptosis in trophoblast cells remains unclear. Since p53 is a critical regulator of apoptosis we hypothesized that increased temperature in placenta produces abnormal expression of proteins in the p53 pathway and finally caspase-3 activation. Moreover, leptin, produced by placenta, is known to promote the proliferation and survival of trophoblastic cells. Thus, we aimed to study the possible role of leptin preventing apoptosis triggered by high temperature, as well as the molecular mechanisms underlying this effect. Fresh placental tissue was collected from normal pregnancies. Explants of placental villi were exposed to 37 °C, 40 °C and 42 °C during 3 h in the presence or absence of 10 nM leptin in DMEM-F12 medium. Western blotting and qRT-PCR was performed to analyze the expression of p53 and downstream effector, P53AIP1, Mdm2, p21, BAX and BCL-2 as well as the activated cleaved form of caspase-3 and the fragment of cytokeratin-18 (CK-18) cleaved at Asp396 (neoepitope M30). Phosphorylation of the Ser 46 residue on p53, the expression of P53AIP1, Mdm2, p21, as well as caspase-3 and CK-18 were significantly increased in explants at 40 °C and 42 °C. Conversely, these effects were significantly attenuated by leptin 10 nM at both 40 °C and 42 °C. The BCL2/BAX ratio was also significantly decreased in explants at 40 °C and 42 °C compared with explants incubated at 37 °C, which was prevented by leptin stimulation. These data illustrate the potential role of leptin for reducing apoptosis in trophoblast explants, including trophoblastic cells, triggered by high temperature, by preventing the activation of p53 signaling.

Regulation of leptin expression by 17beta-estradiol in human placental cells involves membrane associated estrogen receptor alpha.

The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17ß-estradiol (E(2)) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E(2) conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E(2) also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E(2) induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E(2) genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E(2) regulates leptin expression in placenta and support the importance of leptin in placental physiology.

Leptin receptor activation increases Sam68 tyrosine phosphorylation and expression in human trophoblastic cells.

Leptin is produced in placenta where it has been found to be an important autocrine signal for trophoblastic growth during pregnancy, promoting antiapoptotic and trophic effects. Leptin receptor is present in trophoblastic cells and leptin may fully activate signaling. We have previously implicated the RNA-binding protein Sam68 in leptin signal transduction in immune cells. In the present work, we have studied the possible role of Sam68 in leptin receptor signaling in trophoblastic cells (JEG-3 cells). Leptin dose-dependently stimulated Sam68 phosphorylation in JEG-3 cells, as assessed by immunoprecipitation and immunoblot with anti-phosphotyrosine antibodies. As previously observed in other systems, tyrosine phosphorylation of Sam68 in response to leptin inhibits its RNA binding capacity. Besides, leptin stimulation dose-dependently increases Sam68 expression in JEG-3 cells, as assessed by quantitative PCR. Consistently, the amount of Sam68 protein is increased after 24h of leptin stimulation of trophoblastic cells. In order to study the possible role of Sam68 on leptin receptor synthesis, we employed antisense strategy to knockdown the expression of Sam68. We have found that a decrease in Sam68 expression leads to a decrease in leptin receptor amount in JEG-3 cells, as assessed both by quantitative PCR and immunoblot. These results strongly suggest the participation of Sam68 in leptin receptor signaling in human trophoblastic cells, and therefore, Sam68 may mediate some of the leptin effects in placenta.

Regulation of placental leptin expression by cyclic adenosine 5'-monophosphate involves cross talk between protein kinase A and mitogen-activated protein kinase signaling pathways.

Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu)(2)cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 microM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)(2)cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 microm PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)(2)cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 microm PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways.

17Beta-estradiol enhances leptin expression in human placental cells through genomic and nongenomic actions.

The process of embryo implantation and trophoblast invasion is considered the most limiting factor in the establishment of pregnancy. Leptin was originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, it has been suggested that leptin is involved in other functions during pregnancy, particularly in the placenta, where it was found to be expressed. In the present work, we have found a stimulatory effect of 17beta-estradiol (E(2)) on endogenous leptin expression, as analyzed by Western blot, in both the BeWo choriocarcinoma cell line and normal placental explants. This effect was time and dose dependent. Maximal effect was achieved at 10 nM in BeWo cells and 1 nM in placental explants. The E(2) effects involved the estrogen receptor, as the antagonist ICI 182 780 inhibited E(2)-induced leptin expression. Moreover, E(2) treatment enhanced leptin promoter activity up to 4-fold, as evaluated by transient transfection with a plasmid construction containing the leptin promoter region and the reporter gene luciferase. This effect was dose dependent. Deletion analysis demonstrated that a minimal promoter region between -1951 and -1847 bp is both necessary and sufficient to achieve E(2) effects. Estradiol action involved estrogen receptor 1, previously known as estrogen receptor alpha, as cotransfection with a vector encoding estrogen receptor 1 potentiated the effects of E(2) on leptin expression. Moreover, E(2) action probably involves membrane receptors too, as treatment with an estradiol-bovine serum albumin complex partially enhanced leptin expression. The effects of E(2) could be blocked by pharmacologic inhibition of MAPK and the phosphoinositide-3-kinase (PI3K) pathways with 50 microM PD98059 and 0.1 microM Wortmannin, respectively. Moreover, cotransfection of dominant negative mutants of MAP2K or MAPK blocked E(2) induction of leptin promoter. On the other hand, E(2) treatment promoted MAPK1/MAPK3 and AKT phosphorylation in placental cells. In conclusion, we provide evidence suggesting that E(2) induces leptin expression in trophoblastic cells, probably through genomic and nongenomic actions via crosstalk between estrogen receptor 1 and MAPK and PI3K signal transduction pathways.

Up-regulation of placental leptin by human chorionic gonadotropin.

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.

Leptin promotes cell proliferation and survival of trophoblastic cells.

Leptin, the 16-kDa protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by (3)H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 microM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 microM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation.

Induction of p19INK4d in response to ultraviolet light improves DNA repair and confers resistance to apoptosis in neuroblastoma cells.

The genetic instability driving tumorigenesis is fueled by DNA damage and by errors made by the DNA replication. Upon DNA damage the cell organizes an integrated response not only by the classical DNA repair mechanisms but also involving mechanisms of replication, transcription, chromatin structure dynamics, cell cycle progression, and apoptosis. In the present study, we investigated the role of p19INK4d in the response driven by neuroblastoma cells against DNA injury caused by UV irradiation. We show that p19INK4d is the only INK4 protein whose expression is induced by UV light in neuroblastoma cells. Furthermore, p19INK4d translocation from cytoplasm to nucleus is observed after UV irradiation. Ectopic expression of p19INK4d clearly reduces the UV-induced apoptosis as well as enhances the cellular ability to repair the damaged DNA. It is clearly shown that DNA repair is the main target of p19INK4d effect and that diminished apoptosis is a downstream event. Importantly, experiments performed with CDK4 mutants suggest that these p19INK4d effects would be independent of its role as a cell cycle checkpoint gene. The results presented herein uncover a new role of p19INK4d as regulator of DNA-damage-induced apoptosis and suggest that it protects cells from undergoing apoptosis by allowing a more efficient DNA repair. We propose that, in addition to its role as cell cycle inhibitor, p19INK4d is involved in maintenance of DNA integrity and, therefore, would contribute to cancer prevention.

Inhibitory effect of AP-1 complex on 5-aminolevulinate synthase gene expression through sequestration of cAMP-response element protein (CRE)-binding protein (CBP) coactivator.

Activation protein-1 (AP-1) transcription factors are early response genes involved in a diverse set of transcriptional regulatory processes. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is often used to induce AP-1 activity. The purpose of this work was to explore the molecular mechanisms involved in the TPA regulation of ubiquitous 5-aminolevulinate synthase (ALAS) gene expression, the first and rate-controlling step of the heme biosynthesis. Previous analysis of the 5'-flanking sequence of ALAS revealed the existence of two cAMP-response elements (CRE) required for basal and cAMP-stimulated expression. The fragment -833 to +42 in the 5'-flanking region of rat ALAS gene was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector pALAS/CAT produced a significant CAT activity in transiently transfected HepG2 human hepatoma cells, which was repressed by TPA. Sequence and deletion analysis detected a TPA response element (TRE), located between -261 and -255 (TRE-ALAS), that was critical for TPA regulation. We demonstrated that c-Fos, c-Jun, and JunD are involved in TPA inhibitory effect due to their ability to bind TRE-ALAS, evidenced by supershift analysis and their capacity to repress promoter activity in transfection assays. Repression of ALAS promoter activity by TPA treatment or Fos/Jun overexpression was largely relieved when CRE protein-binding protein or p300 was ectopically expressed. When the TRE site was placed in a different context with respect to CRE sites, it appeared to act as a transcriptional enhancer. We propose that the decrease in ALAS basal activity observed in the presence of TPA may reflect a lower ability of this promoter to assemble the productive pre-initiation complex due to CRE protein-binding protein sequestration. We also suggest that the transcriptional properties of this AP-1 site would depend on a spatial-disposition-dependent manner with respect to the CRE sites and to the transcription initiation site.