Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages

The adrenal cortex is a critical steroidogenic endocrine tissue, generated at least in part from the coelomic epithelium of the urogenital ridge. Neither the intercellular signals that regulate cortical development and maintenance nor the lineage relationships within the adrenal are well defined. We have explored adrenal Shh activity and found that Shh is expressed in relatively undifferentiated steroidogenic cells, which signal to the overlying capsule and subjacent nonsteroidogenic mesenchyme cells that we also find are progenitors of steroidogenic lineages. Shh-expressing cells also generate all steroidogenic cell types, but not nonsteroidogenic ones. Shh mutant adrenals have a thin capsule and small cortex. Our findings both support a novel dual lineage, Shh-independent and Shh-dependent, model of adrenocortical development, and identify distinct populations of adrenocortical progenitor and candidate stem cells.     

Re-expression of senescent markers in deinduced reversibly immortalized cells

We have developed a simian virus 40 (SV40) T-antigen immortalized human cell line, 1MR90-D305.2H4 (IDH4), in which the expression of T-antigen is controlled by the mouse mammary tumor virus (MMTV) promoter and thus regulated by steroids such as dexamethasone. Studies on the regulation of proliferation by T-antigen led to the formulation of a two-stage model for human cell immortalization, in which a mortality stage 1 mechanism (M1) was the target of T-antigen action, and an independent mortality stage 2 mechanism (M2) produced crisis and prevented T-antigen from directly immortalizing cells. Rarely, a cell expressing T-antigen escaped crisis (e.g., M2) and was capable of indefinite proliferation. This model predicted that the deinduction of T-antigen in IDH4 cells would lead to the reexpression of the M1 mechanism, and thus a reexpression of the senescent phenotype. Our study confirms the prediction that, in the absence of steroids, IDH4 cells express a variety of morphological and biochemical markers characteristic of normal senescent human fibroblasts.     

Prostaglandin synthetase activity in bovine adrenocortical cells and subcellular preparations: Effect of ACTH

Prostaglandin (PGE₂, PGF_2α) production by bovine fasciculo-reticulata adrenocortical cell suspensions was examined using specific radioimmunoassay procedures. No detectable PGs (> 50 pg) could be measured in the extract from up to 2 × 10⁶ cell incubations after 1 h, with or without the presence of ACTH, although these cells expressed full steroidogenic capabilities under these conditions. The same preparations could produce PGs when supplemented with arachidonic acid but ACTH had no effect on this process. These negative findings could not be explained by analytical artifacts or metabolic transformation. However, an active PG synthetase system was characterized in bovine adrenocortical subcellular preparations. This system converted arachidonate and endogenous substrate(s) to PGE₂ as the major product. No thromboxane or prostacyclin pathways were detected even at high enzyme/substrate ratio. Although the microsornal adrenal cortex PG synthetase activity shares many features with those observed in other tissues (K_m = 8.3 × 10^?5 M, optimal pH at 8.0, stimulation of PGE₂ formation in the presence of glutathione and L-epinphrine), its specific activity was comparatively low (V_max = 2.5 ng PGE₂/min/mg microsornal proteins), which may explain our negative findings using cell suspensions. These findings do not provide evidence to support the hypothesis proposing a role of endogenous PGs in the mechanism of acute ACTH action in the case of bovine adrenal cortex.     

Control of glycosaminoglycan metabolism by ACTH in bovine adrenocortical cells in primary culture

Bovine adrenocortical cells in primary culture actively incorporated [35S]sodium sulphate and [3H]glucosamine into glycosaminoglycans (GAGs). Most of the synthesized GAGs were found associated with the pericellular material and secreted in the culture medium, while less than 15% remained associated with the cell pellet. In all these fractions the major product was identified by its molecular properties and selective degradation procedures as a heparan sulphate structure. Exposure of the cells to ACTH induces a shift of the synthesized GAGs from the cellular to the pericellular compartment, an increase in the heparan sulphate labelling and the occurrence of an additional product characterized as a hyaluronate. These data suggest that GAG metabolism of the adrenocortical cell may be under hormonal control and open the possibility of a modulation of cell activity through modifications of its GAG components, especially those involved in the cell matrix architecture.     

Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions

Background

Research on mesenchymal stromal cells has created high expectations for a variety of therapeutic applications. Extensive propagation to yield enough mesenchymal stromal cells for therapy may result in replicative senescence and thus hamper long-term functionality in vivo. Highly variable proliferation rates of mesenchymal stromal cells in the course of long-term expansions under varying culture conditions may already indicate different propensity for cellular senescence. We hypothesized that senescence-associated regulated genes differ in mesenchymal stromal cells propagated under different culture conditions.

Design and Methods

Human bone marrow-derived mesenchymal stromal cells were cultured either by serial passaging or by a two-step protocol in three different growth conditions. Culture media were supplemented with either fetal bovine serum in varying concentrations or pooled human platelet lysate.

Results

All mesenchymal stromal cell preparations revealed significant gene expression changes upon long-term culture. Especially genes involved in cell differentiation, apoptosis and cell death were up-regulated, whereas genes involved in mitosis and proliferation were down-regulated. Furthermore, overlapping senescence-associated gene expression changes were found in all mesenchymal stromal cell preparations.

Conclusions

Long-term cell growth induced similar gene expression changes in mesenchymal stromal cells independently of isolation and expansion conditions. In advance of therapeutic application, this panel of genes might offer a feasible approach to assessing mesenchymal stromal cell quality with regard to the state of replicative senescence.     

SV40LT基因重组腺病毒载体的包装、鉴定及用于转染日本血吸虫童虫细胞的研究

目的构建携带SV40LT基因的重组腺病毒表达载体,制备具有感染力的重组腺病毒,观察其转染日本血吸虫（Schistosoma japonicum,Sj）童虫细胞后的表达情况。方法采用体外连接法构建好的携带SV40LT基因的重组腺病毒质粒（AdHu5-SV40LT）转化Stbl2感受态菌,获得重组腺病毒质粒后,经Pac I酶切线性化后转染293A细胞,获得出重组腺病毒（AdHu5-SV40LT）。将重组腺病毒感染Sj童虫细胞,采用RT-PCR和免疫组织化学检测SV40LT基因的表达情况。结果重组腺病毒载体质粒可转染293A细胞并可在293A细胞内进行有效的复制;提取病毒DNA进行PCR检测证实含有SV40LT目的基因。以重组腺病毒能感染Sj童虫细胞,经RT-PCR和免疫组化检测有SV40LT基因在细胞中的表达。结论成功包装了具有感染能力的含SV40LT基因的重组腺病毒,感染Sj童虫细胞后目的基因有表达,为进一步探索日本血吸虫细胞永生化提供了实验依据。     

The gene expression profile of psoralen plus UVA-induced premature senescence in skin fibroblasts resembles a combined DNA-damage and stress-induced cellular senescence response phenotype

After a finite number of population doublings, normal human cells undergo replicative senescence accompanied by growth arrest. We previously described a model of stress-induced premature senescence by treatment of dermal fibroblasts with psoralen plus UVA, a common photodermatological therapy. Psoralen photoactivation has long been used as a therapy for hyperproliferative skin disorders. The repetitive therapeutical treatment is accompanied by premature aging of the skin. Treatment of fibroblasts in vitro with 8-methoxypsoralen (8-MOP) and subsequent ultraviolet A (UVA) irradiation results in growth arrest with morphological and functional changes reminiscent of replicative senescence. For gene expression profiling in two strains of human skin fibroblasts after PUVA treatment, we used a low-density DNA array representing 240 genes involved in senescence and stress response. Twenty-nine genes were differentially expressed after PUVA treatment in the two strains of human skin fibroblasts. These genes are involved in growth arrest, stress response, modification of the extracellular matrix and senescence. This study contributes further to the elucidation of the PUVA model and its validation as a useful stress-induced premature senescence model aiming to characterize the premature senescence of fibroblasts and to identify biomarkers that could be applied in vivo.     

MORF4基因诱导HeLa细胞衰老

目的建立MORF4基因高表达诱导HeLa细胞衰老模型,探索MORF4基因对HeLa细胞衰老的影响。方法分别将pcDNA3.1(+)/Flag-MORF4和pcDNA3.1(+)空载质粒转染人宫颈癌HeLa细胞株。SA-β-Gal染色检测细胞衰老,MTT法及流式细胞术检测细胞周期变化及细胞的增殖活力,两者共同确定MG-132处理的最适条件。Western印迹检测MORF4、PCNA基因的表达。结果质粒经酶切和测序鉴定,证明pcD-NA3.1(+)/Flag-MORF4质粒中含有目的基因序列;经浓度梯度1.25、2.5、5、10μmol/L MG-132处理转染细胞2、4、24、48 h后,SA-β-Gal染色和MTT检测结果显示10μmol/L MG-132处理24 h的转染细胞明显衰老,细胞活力降低;细胞周期被阻滞在G0/G1期,S期细胞明显减少,增殖受到抑制;Western印迹检测结果显示MORF4蛋白在细胞中的含量明显升高,而与增殖相关基因PCNA表达下调。结论构建的pcDNA3.1(+)/Flag-MORF4质粒在HeLa细胞中表达;并在MG-132作用下,使MORF4基因的表达产物积累,从而影响PCNA蛋白的表达量,抑制HeLa细胞的增殖活性,阻滞细胞周期的进行,导致细胞进入衰老状态。     

On the specificity of the inhibitory effect of trilostane and aminoglutethimide on adrenocortical steroidogenesis in guinea pig

The effect of trilostane and aminoglutethimide on steroidogenesis was studied on isolated guinea pig adrenocortical cells in order to verify whether, in addition to the inhibitory influence of trilostane on 3β-hydroxysteroid dehydrogenase and isomerase and the inhibition by aminoglutethimide of pregnenolone formation, these inhibitors may also affect other enzymatic steps of Cortisol synthesis. While trilostane completely abolished cortisol production in response to ACTH with concomitant enhancement in pregnenolone and 17-hydroxypregnenolone formation, the conversion of progesterone, 17-hydroxyprogesterone and 11-deoxycortisol into cortisol was not affected by the presence of the inhibitor. Contrasting with this specific inhibitory effect of trilostane, aminoglutethimide inhibited not only pregnenolone formation but also several other enzymatic steps involved in the conversion of this precursor of steroidogenesis into cortisol. Among these additional effects of aminoglutethimide on steroidogenesis, the inhibition of 11β-hydroxylation was clearly demonstrated.     

Differential regulation of myelin gene expression in SV40 T antigen-transfected rat glioma C6 cells

Rat glioma C6 cells were stably transfected with a pSV3-neo plasmid containing SV40 T antigen gene, and geniticin-resistant transfectants (designated C6T cells) were cloned. The C6T cells grew as well-defined foci of cells showing squamous or irregular morphology. The doubling time for transfected cells was reduced by approximately 40% as compared to control C6 cells. The transfection with T-antigen also affected the expression of genes coding for structural myelin proteins and for myelin-associated enzymes. The steady-state level of proteolipid protein (PLP)-specific mRNA in C6T cells was 44% lower than in parental C6 cells. On the other hand, the transfection upregulated the expression of myelin-associated glycoprotein (MAG) by 153%. The activity of 23cyclic AMP phosphodiesterase (CNP) was increased by approximately 80 % in the C6T cells as compared to untransfected, control cells. The activity of calcium-activated neutral proteinase (CANP) was also significantly elevated in the transfectants by approximately 50% and 220% for millimolar and micromolar form respectively. The results indicate that T antigen affects the expression of myelin genes, although, individual genes appear to be differently regulated implying the existence of several independent regulatory mechanisms.     

Transplantation of bovine adrenocortical cells encapsulated in alginate

Current treatment options for adrenal insufficiency are limited to corticosteroid replacement therapies. However, hormone therapy does not replicate circadian rhythms and has unpleasant side effects especially due to the failure to restore normal function of the hypothalamic–pituitary–adrenal (HPA) axis. Adrenal cell transplantation and the restoration of HPA axis function would be a feasible and useful therapeutic strategy for patients with adrenal insufficiency. We created a bioartificial adrenal with 3D cell culture conditions by encapsulation of bovine adrenocortical cells (BACs) in alginate (enBACs). We found that, compared with BACs in monolayer culture, encapsulation in alginate significantly increased the life span of BACs. Encapsulation also improved significantly both the capacity of adrenal cells for stable, long-term basal hormone release as well as the response to pituitary adrenocorticotropic hormone (ACTH) and hypothalamic luteinizing hormone-releasing hormone (LHRH) agonist, [D-Trp6]LHRH. The enBACs were transplanted into adrenalectomized, immunodeficient, and immunocompetent rats. Animals received enBACs intraperitoneally, under the kidney capsule (free cells or cells encapsulated in alginate slabs) or s.c. enclosed in oxygenating and immunoisolating βAir devices. Graft function was confirmed by the presence of cortisol in the plasma of rats. Both types of grafted encapsulated cells, explanted after 21–25 d, preserved their morphology and functional response to ACTH stimulation. In conclusion, transplantation of a bioartificial adrenal with xenogeneic cells may be a treatment option for patients with adrenocortical insufficiency and other stress-related disorders. Furthermore, this model provides a microenvironment that ensures 3D cell–cell interactions as a unique tool to investigate new insights into cell biology, differentiation, tissue organization, and homeostasis.Adrenal insufficiency is the failure of adrenocortical cells to produce adequate amounts of glucocorticoids and/or mineralocorticoids. These steroid hormones play a central role in the body’s homeostasis of energy, salt, and fluid; thus, adrenal insufficiency is a potentially life-threatening condition. The most relevant causes of adrenal insufficiency are autoimmune disorders (up to 80%); infectious diseases; hereditary factors; traumatic, metabolic, or neoplastic conditions; or surgical bilateral adrenalectomy, sometimes due to a compulsory therapeutic strategy in the treatment of adrenal tumors or congenital adrenal hyperplasia (CAH).CAH due to 21-hydroxylase deficiency is the most common form of inherited adrenal insufficiency, presenting with clinical symptoms of neuroendocrine perturbations, virilization, and metabolic diseases in later life. Patients may suffer from hypotensive crises, hypoglycemia, acne, and infertility (1, 2). Current options of treatment consisting of replacement therapy with glucocorticoids, mineralocorticoids, and/or androgens can reverse the symptoms only partially, exhibit the unpleasant side effects of inappropriate glucocorticoid substitution, and leave the patients without the diurnal rhythm of glucocorticoid release. Furthermore, adrenomedullary functions, including catecholamine and neuropeptide secretion, also are disrupted (2), which correlates with cardiovascular risks, hypoglycemia, and physical disability in these patients (1, 3).Adrenal gland transplantation could and would be a desirable therapeutic alternative for these patients if it was available and practical (4). Transplanted organs restore and maintain normal hormonal levels, adequately respond to functional demands, and regulate steroid production in response to endogenous and exogenous stimulation, including the circadian rhythm of hormone secretion. However, the application of this strategy is currently extremely limited due to the lack of human donor organs, the surgical difficulties of adrenal transplantation, and the required chronic immunosuppression.For the correction of adrenocortical insufficiency, transplantation of whole adrenal glands might not be mandatory and the transplantation of isolated adrenal cells may be sufficient. An additional advantage of transplantation using isolated cells is the availability of various immunoisolating materials and methods for immune protection of such transplants. Application of these materials not only allows avoidance of chronic immunosuppression but also allows the transplantation of xenogeneic cells (5, 6).Sodium alginate is one of the clinically approved immunoisolating biopolymers; it has already been widely used in a variety of biomedical applications (5–10). Alginates have several unique structural and chemical parameters, appropriate not only for immune isolation but also for the creation of 3D cellular scaffolds that allow artificial organs to function long term in vitro and in vivo (7, 8, 11). The potential of xenogeneic adrenocortical cells to replace adrenal gland function has already been tested in animal models but requires acquisition of immunodeficiency (12, 13). Alginate encapsulation may protect xenogeneic adrenocortical cells from destruction by immunological processes (6). Alternatively, similar to pancreatic islets (9, 14), adrenocortical cells can be transplanted within special oxygenating and immunoisolating devices, thus reducing the risk of an immunological host versus graft response.Creating a long-lasting, immune-isolated, and functional bioartificial adrenal was the main aim of this research. The objectives of our work included testing of primary bovine adrenocortical cells (BACs) as a potential source of cells, defining optimal conditions, and long-term monitoring. Because adrenocortical cells also express receptors for luteinizing hormone-releasing hormone (LHRH), the effect of the LHRH agonist [D-Trp6]LHRH on adrenocortical steroidogenesis in encapsulated BACs (enBACs) was tested. We characterized bioartificial adrenals in vivo and investigated their functionality and efficacy after implantation into bilaterally adrenalectomized rats.     

A single-stage mechanism controls replicative senescence through Sudden Senescence Syndrome

Normal human cells have a finite proliferative potential in vitro. However, some DNA viral proteins, such as SV40 Tg, can alter this and extend the lifespan after which the cells enter crisis, a period when massive cell death occurs. Based on these observations, a two-stage model for cellular senescence has been proposed with a distinct function for each stage. Mortality stage 1 (M1) is hypothesized to cause cell senescence and is activated near the end of the proliferative lifespan, whereas Mortality stage 2 (M2) involves an independent mechanism that causes failure of cell division and crisis. Here, we present experimental evidence demonstrating that inhibition of the onset of Sudden Senescence Syndrome (SSS) by SV40 Tg greatly reduces the appearance of senescent cells in the culture and results in an increase in the population doublings (PD) to that of the number of cell generations (CGs).This is what causes the observed lifespan extension. Our results also provide an explanation for `additional' telomere shortening during this `extended' lifespan. Based on these observations, we suggest that crisis or M2 cannot be considered a `mechanism' controlled by a specific set of genes. Our results do not support the previously proposed two-stage model and indicates SSS as the single, primary mechanism of cell senescence. Several recent findings from other laboratories that support our previously published self-recombination model of the molecular mechanisms that control SSS are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Absence of replicative senescence in cultured cells from the short-lived killifish Nothobranchius furzeri

A major challenge in age research is the absence of short-lived vertebrate model organisms. The turquoise killifish Nothobranchius furzeri has the shortest known lifespan of a vertebrate that can be bred in captivity. The short lived GRZ strain only reaches a maximum age of 3–4 months, whereas other strains (MZM) reach 6–10 months. Most importantly, the short lifespan is associated with typical signs of ageing. To find out more about possible cellular factors that might contribute to the short lifespan and to the difference in lifespan between strains, we analyzed the expression of markers for cellular senescence. Expression of Tp53, Cdkn1a and Cdkn2a/b in skin revealed no change in the short-lived GRZ but increased expression of the cell cycle inhibitors Cdkn1a and Cdkn2a/b in the long-lived MZM strain with age. This suggests that expression of distinct cell cycle inhibitors reflects rather chronological than biological age in N. furzeri. To study the relationship of organismal life span and in vitro life span of cells, we established a primary cell culture model. For both strains we demonstrate here the absence of replicative senescence as analysed by morphology, expression of Cdkn1a and Cdkn2a/b, population doubling times and γH2AFX in long-term and short-term cultured cells. We reason this to be on account of sustained telomerase activity and maintained telomeric length. Hence, we propose that differences in maximum life span of different N. furzeri strains is not reflected by differences in proliferation speed or replicative potential of the respective cultured cells.     

Melatonin protects bone marrow mesenchymal stem cells against iron overload‐induced aberrant differentiation and senescence

Bone marrow mesenchymal stem cells (BMSCs) are an expandable population of stem cells which can differentiate into osteoblasts, chondrocytes and adipocytes. Dysfunction of BMSCs in response to pathological stimuli contributes to bone diseases. Melatonin, a hormone secreted from pineal gland, has been proved to be an important mediator in bone formation and mineralization. The aim of this study was to investigate whether melatonin protected against iron overload‐induced dysfunction of BMSCs and its underlying mechanisms. Here, we found that iron overload induced by ferric ammonium citrate (FAC) caused irregularly morphological changes and markedly reduced the viability in BMSCs. Consistently, osteogenic differentiation of BMSCs was significantly inhibited by iron overload, but melatonin treatment rescued osteogenic differentiation of BMSCs. Furthermore, exposure to FAC led to the senescence in BMSCs, which was attenuated by melatonin as well. Meanwhile, melatonin was able to counter the reduction in cell proliferation by iron overload in BMSCs. In addition, protective effects of melatonin on iron overload‐induced dysfunction of BMSCs were abolished by its inhibitor luzindole. Also, melatonin protected BMSCs against iron overload‐induced ROS accumulation and membrane potential depolarization. Further study uncovered that melatonin inhibited the upregulation of p53, ERK and p38 protein expressions in BMSCs with iron overload. Collectively, melatonin plays a protective role in iron overload‐induced osteogenic differentiation dysfunction and senescence through blocking ROS accumulation and p53/ERK/p38 activation.