Combined transcriptome studies identify AFF3 as a mediator of the oncogenic effects of ß-catenin in adrenocortical carcinoma.

Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/ß-catenin signaling pathway. However, the adrenal-specific targets of oncogenic ß-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous ß-catenin activating mutation was done to identify the Wnt/ß-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that AFF3 mediates the oncogenic effects of ß-catenin in ACC. The Wnt response element site located at nucleotide position -1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/ß-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/ß-catenin pathway involved in ACC, acting on transcription and RNA splicing.

Comparison of the effects of PRKAR1A and PRKAR2B depletion on signaling pathways, cell growth, and cell cycle control of adrenocortical cells.

The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression.

Protein kinase A alterations in endocrine tumors.

Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis.

1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase.

Lung maturation before birth includes type II pneumocyte differentiation with progressive disappearance of glycogen content and onset of surfactant synthesis. We have shown previously that 1,25-(OH)2D3 increases surfactant synthesis and secretion by type II cells and decreases their glycogen content in fetal rat lung explants. Recently, the gene coding fructose 1,6 bisphosphatase (F1,6BP), a regulatory enzyme of gluconeogenesis, has been identified in type II cells and its promoter bears a Vitamin D response element. Present results show:The coexistence of type II cells at different stages of maturation. in rat fetal lung on day 21 of gestation (electron microscopy), and the association between maturation of type II cells and disappearance of their glycogen content. The immunogold labeling of all type II cells when using the 9A7g VDR-antibody, with significantly more abundant gold particles in cells exhibiting an intermediate glycogen content. The expression of F1,6BP mRNA in a human type II cell line (NCI-H441) and the increase of this expression after 18h incubation with 1,25-(OH)2D3 (10(-8)M). These results bring further evidence for a physiological role of 1,25-(OH)2D3 during type II pneumocyte maturation. Activation of F1,6BP may participate to the 1,25-(OH)2D3 action on surfactant synthesis via the gluconeogenesis pathway.

Tryptophan missense mutation in the ligand-binding domain of the vitamin D receptor causes severe resistance to 1,25-dihydroxyvitamin D.

In this study, two related young children, brother and sister, exhibited severe vitamin D-resistant rickets without alopecia. Sequence analysis of the total vitamin D receptor (VDR) cDNA from skin fibroblasts revealed a substitution of the unique tryptophan of the VDR by arginine at amino acid 286 (W286R). Cultured skin fibroblasts of the two patients expressed normal-size VDR protein (immunocytochemistry and Western blotting) and normal length VDR mRNA (Northern blotting). But, these fibroblasts, as well as COS-7 cells transfected with the W286R mutant, failed to bind 3H 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The tryptophan substitution did not affect VDR trafficking toward the nucleus but abolished the 24-hydroxylase gene response to 1,25(OH)2D3, even at 10(-6) M concentrations. In conclusion, this case report of a new family with hereditary vitamin D-resistant rickets (HVDRR) emphasizes the crucial role of the VDR tryptophan for ligand binding and for transactivation of 1,25(OH)2D3 target genes. It clearly shows the clinical significance of this VDR amino acid for calcium homeostasis and bone mineralization. This observation suggests further that the presence of a stable VDR-bound ligand may not be obligatory for normal hair follicle development.

In vivo effects of vitamin D on the proliferation and differentiation of rat keratinocytes.

The hormonal form of vitamin D appears to be a physiological regulator of the epidermogenesis. While its differentiation-promoting effect is well accepted, there are conflicting reports of its action on keratinocyte proliferation. This study evaluates the specific changes induced by vitamin D treatment in the epidermis of rats nutritionally deprived of vitamin D by cell size analysis, acridine orange flowcytometry, and the immunohistochemical detection of proteins related to the different stages of differentiation (epidermal calcium binding protein and suprabasal keratins recognized by KL1 antibody) The total keratinocyte and isolated keratinocyte subpopulations were studied. Vitamin D deficiency was associated in the total population with a lower percentage of actively proliferating cells and with a lack of differentiation markers. Study of the isolated cell populations demonstrated, however, that small cells were actively proliferating, whereas they were mainly in the resting stage in the normal epidermis. Treatment with vitamin D dramatically increased cell proliferation and stimulated the appearance of differentiation markers. Some of the observed effects, such as an increase in proliferation and the appearance of epidermal calcium binding protein, were due to the normalisation of the vitamin D deficiency-induced hypocalcemia, whereas the expression of suprabasal keratins was directly dependent on vitamin D. We conclude that the action of vitamin D on the epidermis is associated with increases in both proliferation and differentiation of keratinocytes. Vitamin D itself and its resulting action on calcium homeostasis appear to contribute to the observed effects.

Resistance to murine AIDS in offspring of mice infected with LP-BM5. Role of CD8 T cells.

The murine-acquired immunodeficiency syndrome (MAIDS) is caused by a mixture of murine leukemia viruses (LP-BM5 MuLV). The influence of perinatal contact with retroviruses or their Ags on the response to infection was tested by infecting with LP-BM5 (MuLV) the adult offspring of mice with MAIDS. These offspring were resistant to disease after virus challenge. Most of them were free of defective viral DNA, and even those with molecular evidence of infection had lymphoid cells with a lower infectious capacity to cause MAIDS in naive recipients. No ecotropic, xenotropic, or mink cell focus-forming (MCF) virus expression was found at the age of 5 wk, which is the time of LP-BM5 (MuLV) challenge. However, at 22 wk of age, one-half of the offspring from MAIDS mothers had ecotropic virus-expressing cells in their spleens. At the time of suckling, offspring from infected mothers had enhanced percentages of B cells and CD4 and CD8 T cells in the spleen, possibly followed by a slight persistent splenomegaly. These results suggest that immune reactivity, rather than tolerance to the virus, is responsible for resistance to disease after challenge. The offspring of MAIDS mice could clear the virus after challenge. This clearance was mediated by CD8 T cells, as continuous CD8 T cell depletion initiated at the time of viral challenge abrogated the resistance of these mice to MAIDS.

Synthesis of and response to 1,25 dihydroxycholecalciferol by subpopulations of murine epidermal keratinocytes: existence of a paracrine system for 1,25 dihydroxycholecalciferol.

The epidermis is both a target tissue for and a source of 1,25 dihydroxycholecalciferol. The present study determines which of the epidermal cell populations synthesizes 1,25 dihydroxycholecalciferol and which responds to this hormone. Epidermal keratinocytes from new born rat epidermis were separated by unit gravity sedimentation into poorly differentiated cells, slow-cycling more differentiated cells, actively proliferating cells, and terminally differentiating subpopulations. The keratinocyte populations were characterized by cell size analysis, cell morphology, and DNA and RNA contents (acridine orange flow cytometry). 1,25(OH)2D3 synthesis was studied by measuring the conversion of [3H] 25(OH)D3 to [3H] 1,25(OH)2D3. The purified product was tested for its ability to compete with synthetic [3H] 1,25(OH)2D3 for binding to chick intestinal cytosol. The responses of the keratinocyte subpopulations to exogenous 1,25(OH)2D3 were evaluated by the increase in 25(OH)D3-24 hydroxylase activity. Furthermore the expression of 1,25(OH)2D3 receptors (VDR) was examined in these cell populations. The results show that only the least differentiated cells produced 1,25(OH)2D3. In contrast, immunocytochemical detection of VDR, the VDR mRNA, and a 25(OH)D3-24 hydroxylase response to 1,25(OH)2D3 were mainly found in the more differentiated cells. Thus, the ability of epidermis to synthesize 1,25(OH)2D3 and be simultaneously sensitive to it depends on the state of cell differentiation. This suggests that the mammalian epidermis contains a paracrine system in which the more differentiated keratinocytes are sensitive to the 1,25(OH)2D3 produced locally by neighboring immature ones.

The significance of the pre-challenge immune status of mice for development of retrovirus-induced immunodeficiency syndrome (MAIDS).

The effects of vaccination with RNA-free viral pseudoparticles, preinfection with non-pathogenic ecotropic virus, and induction of tolerance to viral proteins in newborns on the outcome of murine immunodeficiency syndrome (MAIDS) were studied. The parameters used to follow disease progression were: lymphopenia, circulating B and T8 cells, serum IgG and IgM levels, lymphoproliferation and skin graft rejection. Immunization with RNA-free viral pseudoparticles had no effect on any of these parameters. Preinfection of adults with ecotropic virus and the induction of tolerance in newborns to virus antigens both attenuated the early symptoms of viral infection and delayed the onset of immunodeficiency and lymphoproliferation in some mice, but did not significantly alter the number of deaths due to MAIDS. Failure of immune-based therapy to produce successful protection against MAIDS suggests that immune destruction caused by the persistent virus rather than hyperimmune activity is the main pathogenic factor in this disease.

Epidermal calcium-binding protein: a marker of early differentiation of basal layer keratinocytes of rats.

Epidermal calcium-binding protein (ECaBP) is present in the cells of the basal layer of the epidermis and other stratified epithelia. Since the basal layer compartment contains at least two types of cells: slow-cycling, poorly-differentiated, and actively proliferating, more differentiated cells, it was of interest to determine whether they both contained ECaBP. Basal and nearly suprabasal layer keratinocytes from newborn rat epidermis were fractionated into three fractions on the basis of cell size, using low-gravity sedimentation. The cell differentiation in each subgroup was estimated by cell size, morphology, cell cycle stage, RNA/DNA content, and the presence of specific keratins. The presence of ECaBP in these fractions was detected by immunocytochemistry and immunoblotting. Double staining with ECaBP antibodies and propidium iodide followed by flow cytometry was used to correlate ECaBP production and the stage of cell cycle. The relative cell size, measured by the light scattering was used to study the relationship between cell size and ECaBP production. The results show that small keratinocytes with low DNA and RNA content (G0 cells) do not express ECaBP. ECaBP was found only in intermediate size basal keratinocytes with higher DNA and RNA contents, corresponding to actively proliferating S phase cells. Large keratinocytes, which express suprabasal keratin and have low DNA and high RNA content, cease to express ECaBP. ECaBP may, therefore, be a useful marker for assessing the movement of cells from poorly differentiated reserve compartment towards proliferation and further differentiation in both physiological and pathological situations.

Characteristics of homogeneously small keratinocytes from newborn rat skin: possible epidermal stem cells.

The aims of the present study were to characterize the phenotype, growth kinetics, and proliferative activation in culture of a population of poorly differentiated homogeneously small (HS) keratinocytes. These slow-cycling cells were separated by unit gravity sedimentation from a population of actively proliferating basal keratinocytes in newborn rat skin. This population (approximately 1% of the total basal keratinocytes) consisted of extremely small cells with little cytoplasm or RNA. Their positive KL4 staining demonstrates that they were keratinocytes. HS keratinocytes did not, however, contain epidermal calcium binding protein. Acridine orange, bivariate Hoechst, and ethidium bromide flow cytometry of in vitro bromodeoxyuridine-labeled cells as well as Ki67 staining showed that HS keratinocytes were in the G0 stage of the cell cycle and did not actively proliferate in vivo. [3H]thymidine label-retaining cells were found only in the HS cell population, showing that HS cells may originate from a central position in the epidermal proliferative unit. Growth of HS cells in vitro was characterized by a delayed but progressive increase in RNA before entry into the cell cycle. The clonogenic efficiency of HS cells in primary culture was much less than that of larger cells. Subclones of HS cell colonies exceeded primary colonies in their cloning efficiency and proliferative potential, suggesting that HS cells, although normally prevented from dividing, retain a high self-renewal capacity. They also maintain the ability to differentiate. The results are consistent with the concept that HS cell population may represent the epidermal-specific progenitor cells which act as stem cells in this tissue.

Cell size and RNA content correlate with cell differentiation and proliferative capacity of rat keratinocytes.

Keratinocytes from rat skin were separated according to their size in a specially designed unit-gravity sedimentation chamber. The fractions obtained with this technique showed clear morphological differences, and analysis of size distribution confirmed that size was the criterion for separation. Simultaneous DNA and RNA staining of the fractions with acridine orange and subsequent flow cytometric analysis enabled one to classify cells into resting, proliferating, and differentiating stages. Cell size was not directly correlated with proliferation in situ as determined with acridine orange flow cytometry, nor with proliferative capacity in culture as assayed by BrdU/Hoechst flow cytometry. The smallest cells, exhibiting low DNA and RNA content, which do not proliferate in vivo, required a prolonged period of serum stimulation in vitro to initiate RNA and DNA synthesis. Cells of intermediate size exhibited early RNA synthesis and maximal proliferative capacity, whereas the largest cell population displayed no RNA synthesis in culture and the least proliferative capacity. In conclusion, these results suggest that RNA synthesis early after serum stimulation, in addition to a specific, optimal cell size, correlates with the proliferative capacity of keratinocytes in cell culture.

Cell subpopulations within proliferative and differentiating compartments of epidermis.

The heterogeneous population of newborn rat keratinocytes was separated into different subgroups according to their cell size. The relation between cell size, position in the cell cycle, RNA content, and proliferative potential in culture was examined. A reserve stem cell population of Go/G1 cells, low in RNA, giving rise to colonies of undifferentiated phenotype in cell culture, has been separated from more differentiated transit basal cells. In the fractions of the larger cells, several subgroups, probably corresponding to different stages of differentiation, were identified: G2M cells with low RNA content, large S-phase cells rich in RNA, and small Go/G1 cells low in RNA. The clonogenic cells from these fractions have limited growth potential and give rise to moderately or terminally differentiated colonies. The selective sorting of stem cell populations may be useful for elucidating the mechanism of carcinogenesis in epidermis and other proliferative tissues. Analysis of the relative proportions of cell subpopulations represents a novel approach leading to the refinement of the concepts of epidermal structure in physiological and pathological states. It also could, by extension, shed new light on the behavior of other proliferative tissues.

Effect of vitamin D deficiency and 1,25-dihydroxycholecalciferol treatment on epidermal calcium-binding protein (ECaBP) RNA activity.

We have previously reported that the amount of epidermal calcium binding protein (ECaBP) in the skin decreases in the absence of vitamin D. Since vitamin D influences epidermal differentiation, and the synthesis of ECaBP may vary with cell differentiation, it was necessary to know whether vitamin D acts directly on the translational or post-translational level of ECaBP synthesis or indirectly by its action on epidermopoiesis. The cell-free translation technique was used to demonstrate the presence of mRNA coding for ECaBP. The activity of this mRNA has been evaluated in the skin of vitamin D-fed and in vitamin D-deficient rats with or without treatment with 1,25-dihydroxycholecalciferol (1,25(OH)2D3). Vitamin D deficiency decreased the ECaBP mRNA activity. The latter was selectively increased in animals given a single dose of 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 stimulates the production of ECaBP mRNA or stabilizes this mRNA.

Cell free translation of rat epidermal calcium-binding protein (EP-12) messenger RNA.

The primary step in the biosynthesis of 12 KDa rat epidermal calcium binding protein was studied by cell-free protein synthesis. Poly(A)+ rich RNA was extracted and purified from whole newborn rat skin and translated in a lysate system in the presence of labeled methionine. Immunoprecipitation of translation products with a monospecific antibody directed against this protein, which did not react with parvalbumin yielded a product migrating as a single band of molecular weight 12 KDa on polyacrylamide gel electrophoresis. Thus, a mRNA coding for this protein is present in rat skin. The presence of this messenger RNA opens the way for further studies on the regulation of epidermal expression during epidermal cell proliferation and differentiation.