Daniela Willems

TGF-β autocrine loop regulates cell growth and myogenic differentiation in human rhabdomyosarcoma cells

Transforming growth factor β (TGF) is a well‐known inhibitor of myogenic differentiation as well as an autocrine product of rhabdomyosarcoma cells. We studied the role of the TGF‐β autocrine loop in regulating growth and myogenic differentiation in the human rhabdomyosarcoma cell line, RD. We previously reported that the phorbol ester 12‐O‐tetradeca‐noylphorbol‐13‐acetate (TPA) induces growth arrest and myogenic differentiation in these cells, which constitutively express muscle regulatory factors. We show that TPA inhibits the activation of secreted latent TGF‐β, thus decreasing the concentration of active TGF‐β to which the cells are exposed. This event is mediated by the TPA‐induced alteration of the uPA/ PAI serine‐protease system. Complete removal of TGF‐β, mediated by the ectopic expression of a soluble type II TGF‐β receptor dominant negative cDNA, induces growth arrest, but does not trigger differentiation. In contrast, a reduction in the TGF‐β concentration, to a range of 0.14–0.20 × 10−2 ng/ml (which is similar to that measured in TPA‐treated cells), mimics TPA‐induced differentiation. Taken together, these data demonstrate that cell growth and suppression of differentiation in rhabdomyosarcoma cells require overproduction of active TGF‐β; furthermore, they show that a ‘critical’ concentration of TGF‐β is necessary for myogenic differentiation to occur, whereas myogenesis is abolished below and above this concentration. By impairing the TGF‐β autocrine loop, TPA stabilizes the factor concentration within the range compatible for differentiation to occur. In contrast, in human primary muscle cells a much higher concentration of exogenous TGF‐β is required for the differentiation inhibitory effect and TPA inhibits differentiation in these cells probably through a TGF‐β independent mechanism. These data thus clarify the mechanism underlying the multiple roles of TGF‐β in the regulation of both the transformed and differentiated phenotype.—Bouche, M., Canipari, R., Mel‐chionna, R., Willems, D., Senni, M. I., Molinaro, M. TGF‐β autocrine loop regulates cell growth and myo‐genic differentiation in human rhabdomyosarcoma cells. FASEB J. 14, 1147–1158 (2000)

In Vivo and in Vitro Experimental Analysis of Lens Regeneration in Larval Xenopus laevis

After lentectomy of larval Xenopus laevis, the outer cornea undergoes tissue transformation resulting in formation of a new lens. This lens regeneration is triggered and sustained by neural retina. In the present study, lens‐forming transformation of the outer cornea was completed in vitro when the outer cornea was cultured within the lentectomized eye‐cup. Well‐differentiated lens fiber cells, which showed positive immunofluorescence for total crystallins, were also formed when the outer cornea was cultivated with the retina. No lens tissue was formed when the cornea was cultured alone. Lens‐forming transformation, originating from the cornea three and five days after lentectomy, completely regressed when the tissue was isolated in vitro. Fom the present and previous findings, we concluded that, the interaction of corneal cells with the retina plays a decisive role in lens regeneration in situ.

Lens fibre transdifferentiation in cultured larval Xenopus laevis outer cornea under the influence of neural retina-conditioned medium

Abstract. The outer cornea of larval Xenopus laevis can reprogram cell differentiation when cultured in medium conditioned by X. laevis neural retina (XRCM) or by Rana esculenta neural retina (RRCM). Under these experimental conditions corneal cells showed the same series of cytological changes of fibre cell differentiation observed during ontogenesis and in vivo lens regeneration enlargement of nuclei and nucleoli, increase of ribosomal population (cytoplasmbasophilia), cell elongation, gradual loss of basophilic properties and acquisi tion of acidophilic properties for crystallin synthesis and accumulation. These events were completely dependent on XRCM or RRCM, suggesting that the neural retina secretes a factor(s) which initiates and sustains lens fibre transdifferentiation of the corneal epithelial cells. This culture system appears to be a suitable one for investigating the control of lens fibre transdifferentiation in vitro.

Persistence of the lens-inducing capacity of the neural retina in adult Anura

In the present research the neural retina of adultXenopus laevis andRana esculenta was implanted into the enucleated orbit of larvalXenopus laevis to ascertain if the lens-inducing power of this ocular tissue was maintained in the post-metamorphic stages. The results obtained show that the inducing power of the neural retina is maintained in the adult eye without significant variations.RiassuntoNella presente ricerca sono stati effettuati esperimenti di impianto di retina neurale diXenopus laevis eRana esculenta adulti nell’orbita enucleata di larve diXenopus laevis con il fine di accertare l’eventuale persistenza del potere induttore lentogeno negli occhi degli Anuri adulti. I risultati ottenuti mosrrano ehe il potere induttore viene mantenuto senza significative variazioni.

Primi risultati di transdifferenziamento lentogeno delta cornea di larve di Xenopus laevis indotto da aFGF

It has been shown that lens regeneration from outer cornea in larvalXenopus laevis is dependent on the influence of neural retina bothin situ and in tissue culture. In the present research the outer cornea of larvalXenopus laevis was cultured for 4–10 days in serum supplemented diluted Leibovitz L 15 added with 500 ng/ml of brain derived acidic FGF. The obtained results show that aFGF can stimulate lens-forming transformation of the outer cornea and suggest that this growth factor may be one of the peptidic factors normally produced by the retina during lens regeneration.RiassuntoPrecedenti ricerche hanno dimostrato che la rigenerazione délia lente dalla cornea esterna di larve di Xenopuslaevis dipende dall’influenza esercitata dalla retina neurale siain situ sia in coltura di tessuto. Nella présente ricerca la cornea esterna di larve diXenopus laevis è stata coltivatain vitro in Leibovitz L 15 diluito, in presenza di siero fetale di vitello e di 500 ng/ml di FGF acido. I risultati ottenuti evidenzian-do che il transdifferenziamento lentogeno della cornea può essere stimolato da aFGF e suggeriscono che tale fattore di crescita potrebbe essere prodotto dalla retina neurale durante la rigenerazione della lente.Precedenti ricerche hanno dimostrato che la rigenerazione delia lente dalla cornea esterna di larve di Xenopuslaevis dipende dall’influenza esercitata dalla retina neurale siain situ sia in coltura di tessuto. Nella presente ricerca la cornea esterna di larve diXenopus laevis e stata coltivatain vitro in Leibovitz L 15 diluito, in presenza di siero fetale di vitello e di 500 ng/ml di FGF acido. I risultati ottenuti evidenzian-do che il transdifferenziamento lentogeno della cornea puo essere stimolato da aFGF e suggeriscono che tale fattore di crescita potrebbe essere prodotto dalla retina neurale durante la rigenerazione della lente.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

BackgroundSuccess of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes.ResultsWe tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and hence presumably of the GDF8 gene, in both CHO and C2C12 cultured cells.ConclusionsIn vitro the MYOD1-A allelic variant could up-regulate the expression of MYOD1 gene. Additionally, we could assess a different response of in vitro gene expression according to cell type used to transfect constructs, suggesting that MyoD activation is regulated by mechanisms that are specific of myoblasts.

Differenziamento di trapianti di epitelio della lente di larve di Xenopus laevis

Nella presente ricerca l’epitelio della lente di larve diXenopus laevis e stato isolato ed impiantato autoplasticamente al fine di accertare la possibile capacita differenziativa autonoma di questo tessuto. I risultati ottenuti mostrano che il differenziamento deu’epitelio della lente e indipendente dalla presenza del’occhio e suggeriscono quindi 1’esistenza di capacita differenziative autonome di questo tessuto oculare.In the present research the lens epithelium of larvalXenopus laevis was isolated and autoplastically implanted into the enucleated orbit to ascertain the autonomous differentiative capacity of this tissue. The results obtained show that the eye cup is not necessary for the differentiation of the lens epithelium into lens fiber cells and suggest an intrinsic lens differentiation capacity of this tissue.RiassuntoNella presente ricerca l’epitelio della lente di larve diXenopus laevis è stato isolato ed impiantato autoplasticamente al fine di accertare la possibile capacità differenziativa autonoma di questo tessuto. I risultati ottenuti mostrano che il differenziamento deü’epitelio della lente è indipendente dalla presenza del’occhio e suggeriscono quindi 1’esistenza di capacità differenziative autonome di questo tessuto oculare.

SNPs identification in swine leptin 5’ flanking region and transcriptional activity of naturally occurring promoter haplotypes

Leptin is an adipocyte-derived hormone which acts as a major regulator for food intake and energy homeostasis. Previous studies have focused on the association between polymorphisms in the coding region of the leptin (LEP) pig gene and economically important traits. The present work aimed to study the leptin gene at the promoter region to search for polymorphisms that could lead to different gene expression regulation. In the 1213 bp 5’ gene flanking region, we identified 17 new polymorphisms in two pig breeds characterized by different fat content, namely Casertana and Large White. In order to investigate whether a polymorphism affects the transcriptional activity of the leptin promoter, we evaluated in vitro reporter gene activity driven by two constructs harboring different naturally occurring haplotypes, and we found a two-fold difference in transcription efficiency (P<0.04). These findings may serve as a basis for future studies even in different breeds and might provide molecular data that can be used in association with phenotypic data and finally in the selection scheme.

Cell cultures harbouring constructs of different pig promoter polymorphisms show different transcriptional efficiency in gene reporter systems

Abstract Production traits variability among and within breeds, differences among developmental stages or the response to different environments are in part due to genetic factors that affect gene expression. Within the context of an Italian FIRB project, whose objective is to identify genes and molecular mechanisms affecting meat quality and production traits in pig, we studied the promoter regions of candidate genes selected on the basis of their physiological role in animal tissue development or composition. Genomic DNA was isolated from liver or muscle tissue of individuals belonging to Large White and Casertana breed. PCR primers were designed to amplify 5’ upstream region of SCD (Stearoyl-CoA Desaturase), LDLR (Low Density Lipoprotein Receptor), LEP (Leptin), MSTN (Myostatin), ACTA1 (Alpha-actin) and HFABP (Heart Fatty Acid Binding Protein) genes using sequences available at NCBI. A total of 19 single nucleotide polymorphisms (SNPs) not previously described were characterised. Some haplotypes, harbouring SNPs located within, or closely flanking potential cis-acting elements, were used to carry out an in vitro analysis of the efficiency of promoter natural variants. Up to 1200 bases upstream the translation start codon were cloned in pGL3 basic vector in phase with the downstream luciferase reporter gene. The different luminescence intensities showed by constructs harbouring allelic variants suggest transcriptional efficiency influenced by polymorphism. The knowledge of a correlation between a different transcriptional activity and a particular haplotype could be a useful tool to identify variation at genes controlling important traits in pig.

In vivo and in vitro experimental analysis of lens epithelium differentiative capacity in Xenopus laevis

Abstract In the present study, the differentiative capacities of Xenopus laevis lens epithelium were tested by isolating it at different stages of development and implanting it autoplastically and homoplastically into the enucleated orbit. The autonomous differentiative capacity of this tissue was also tested in expiants cultured in vitro in Leibovitz L 15 with fetal bovine serum added, or serum‐free. The results obtained from in vivo experiments showed that whatever were the developmental stages, the eye cup was not necessary for differentiation of lens epithelium into lens fiber cells; this suggested that this tissue has an intrinsic capacity to differentiate into lens. Furthermore, lens epithelium showed an autonomous lens differentiation capacity also when isolated and cultured in vitro, with or without addition of fetal bovine serum.