isolation-and-culture-of-mouse-primary-pancreatic-acinar-cells

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Centre de Recherche en Cancérologie de Lyon

Université de Lyon

Université Lyon 1

Centre Léon Bérard

Signaling of TGFbeta family members such as activin is tightly regulated by soluble binding proteins. Follistatin binds to activin A with high affinity, and prevents activin binding to its own receptors, thereby blocking its signaling. We previously identified FLRG gene from a B-cell leukemia carrying a t(11;19)(q13;p13) translocation. We and others have already shown that FLRG, which is highly homologous to follistatin, may be involved in the regulation of the activin function through its binding to activin. In this study, we found that, like follistatin, FLRG protein inhibited activin A signaling as demonstrated by the use of a transcriptional reporter assay, and blocked the activin A-induced growth inhibition of HepG2 cells. We have recently shown that the TGFbeta-induced expression of FLRG occurs at a transcriptional level through the action of Smad proteins. Here we show that activin A increases FLRG and follistatin at both the mRNA and protein levels. We found that Smad proteins are involved in the activin A-induced transcription activation of FLRG and follistatin. Finally we demonstrate that FLRG protein regulates its own activin-induced expression. In conclusion, activin A induces FLRG and follistatin expression. This observation, in conjunction with the antagonistic effect of FLRG and follistatin on activin signaling, indicates that these two proteins participate in a negative feedback loop which regulates the activin function.

Transcription activation of FLRG and follistatin by activin A, through Smad proteins, participates in a negative feedback loop to modulate activin A function.

Activin A, BMP2, and BMP4, members of the TGFbeta family, have been implicated in the regulation of hematopoiesis. Here we explore and compare, for the first time in human primary cells, the role of activin A, BMP2, and BMP4 during erythropoiesis. Using in vitro erythroid differentiation of CD34(+) primary cells, we obtained the main stages of early erythropoiesis, characterized at the molecular, biochemical, and functional levels. Our results indicate that BMP2 acts on early erythroid cells and activin A on a more differentiated population. We report an insight into the mechanism of commitment of erythropoiesis by activin A and BMP2 involving two key events, increase in EPO-R and decrease in GATA2 expression. Simultaneous addition of activin A with BMP molecules suggests that BMP2 and BMP4 differently affect activin A induction of erythropoiesis. Follistatin and FLRG proteins downmodulate the effects of activin A and BMP2 on erythroid maturation.

Regulation of human erythropoiesis by activin A, BMP2, and BMP4, members of the TGFbeta family.

FLRG (follistatin-related gene) is a secreted glycoprotein that is highly homologous with follistatin. These proteins are involved in the regulation of various biological effects mediated by their binding to TGF-beta (transforming growth factor-beta) superfamily members, activin A and bone morphogenetic proteins. To characterize further the function of FLRG, we used a yeast two-hybrid screen to look for other possible functional partners.

FLRG, a new ADAM12-associated protein, modulates osteoclast differentiation.

FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.

A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin.

Follistatin related gene (FLRG) has been previously identified from a chromosomal translocation observed in a B-cell chronic lymphocytic leukemia (B-CLL). FLRG (alternative names: follistatin-related protein, FSRP/follistatin-like-3, FSTL3) is a secreted glycoprotein highly similar to follistatin. Like follistatin, FLRG is involved in the regulation of various biological effects through its binding to members of the transforming growth factor beta (TGFbeta) superfamily such as activin A and myostatin. We have previously shown that TGFbeta and activin A are potent inducers of FLRG transcriptional activation through the Smad proteins. Using a biochemical approach, we investigated whether tumor necrosis factor alpha (TNFalpha) could regulate FLRG expression since TNFalpha plays a critical role in hematopoietic malignancies. We demonstrate that TNFalpha activates FLRG expression at the transcriptional level. This activation depends on a promoter region containing four 107-108 bp DNA repeats, which are evolutionary conserved in primates. These repeats carry a strong phylogenetic signal, which is not common among non-coding sequences. Each DNA repeat contains one TNFalpha responsive element (5'-GGGAGAG/TTCC-3') able to bind nuclear factor kappaB (NF-kappaB) transcription factors. We also show that TGFbeta, through the Smad proteins, potentates the effect of TNFalpha on FLRG expression. This cooperation is unexpected since TGFbeta and TNFalpha usually have opposite biological effects. In all, this work brings new insights in the understanding of FLRG regulation by cytokines and growth factors. It opens attractive perspectives of research that should allow us to better understand the role of FLRG during tumorigenesis.

Identification of NF-kappaB responsive elements in follistatin related gene (FLRG) promoter.

FLRG (follistatin-related gene) is a secreted glycoprotein which is very similar to follistatin. As observed for follistatin, FLRG is involved in the regulation of various biological processes through its binding to members of the TGFbeta (transforming growth factor beta) superfamily, activin, BMPs (bone morphogenetic proteins) and myostatin. Unlike follistatin, FLRG has been found to be both secreted and localized within the nucleus of many FLRG-producing cells, suggesting the existence of specific intracellular functions of the protein.

AF10-dependent transcription is enhanced by its interaction with FLRG.

We generated a transgenic mouse strain (LSL-TbetaRI(CA)) containing a latent constitutively active TGFbeta type I receptor (TbetaRI/ALK5) by using a knock-in strategy into the X chromosome-linked hypoxanthine phosphoribosyl-transferase (Hprt) locus. Transgene expression, under the control of the ubiquitous CAG (human cytomegalovirus enhancer and chicken beta-actin) promoter, is repressed by a floxed transcriptional "Stop" (LSL, Lox-Stop-Lox). In the presence of cre-recombinase, the "Stop" is excised to allow TbetaRI(CA) transgene expression. We showed that restricted expression of TbetaRI(CA) in T lymphocytes efficiently activates TGFbeta signaling and rescues the T-cell autoimmune disorders of TGFbetaRII conditional knockouts. Unexpectedly, our study reveals that TGFbeta signaling upregulation controls T-cell activation but does not impair their development or their peripheral homeostasis. In addition to the information provided on TGFbeta effects on T-cell biology, LSL-TbetaRI(CA) mouse constitutes an attractive tool to address the effect of TGFbeta signaling upregulation in any cell type expressing the cre-recombinase.

Generation of mice with conditionally activated transforming growth factor beta signaling through the TbetaRI/ALK5 receptor.

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-beta controls the differentiation program of iNKT cells. We demonstrate that TGF-beta signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-beta involves the concerted action of different pathways of TGF-beta signaling. Whereas the Tif-1gamma branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1gamma/Smad4-independent branch. Thus, these three different branches of TGF-beta signaling function in concert as complementary effectors, allowing TGF-beta to fine tune the iNKT cell differentiation program.

iNKT cell development is orchestrated by different branches of TGF-beta signaling.

Inactivation of the Transforming Growth Factor Beta (TGFbeta) tumor suppressor pathway contributes to the progression of Pancreatic Ductal AdenoCarcinoma (PDAC) since it is inactivated in virtually all cases of this malignancy. Genetic lesions inactivating this pathway contribute to pancreatic tumor progression in mouse models. Transcriptional Intermediary Factor 1 gamma (TIF1gamma) has recently been proposed to be involved in TGFbeta signaling, functioning as either a positive or negative regulator of the pathway. Here, we addressed the role of TIF1gamma in pancreatic carcinogenesis. Using conditional Tif1gamma knockout mice (Tif1gamma(lox/lox)), we selectively abrogated Tif1gamma expression in the pancreas of Pdx1-Cre;Tif1gamma(lox/lox) mice. We also generated Pdx1-Cre;LSL-Kras(G12D);Tif1gamma(lox/lox) mice to address the effect of Tif1gamma loss-of-function in precancerous lesions induced by oncogenic Kras(G12D). Finally, we analyzed TIF1gamma expression in human pancreatic tumors. In our mouse model, we showed that Tif1gamma was dispensable for normal pancreatic development but cooperated with Kras activation to induce pancreatic tumors reminiscent of human Intraductal Papillary Mucinous Neoplasms (IPMNs). Interestingly, these cystic lesions resemble those observed in Pdx1-Cre;LSL-Kras(G12D);Smad4(lox/lox) mice described by others. However, distinctive characteristics, such as the systematic presence of endocrine pseudo-islets within the papillary projections, suggest that SMAD4 and TIF1gamma don't have strictly redundant functions. Finally, we report that TIF1gamma expression is markedly down-regulated in human pancreatic tumors by quantitative RT-PCR and immunohistochemistry supporting the relevance of these findings to human malignancy. This study suggests that TIF1gamma is critical for tumor suppression in the pancreas, brings new insight into the genetics of pancreatic cancer, and constitutes a promising model to decipher the respective roles of SMAD4 and TIF1gamma in the multifaceted functions of TGFbeta in carcinogenesis and development.

Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas.

Transcriptional intermediary factor 1Î³ (TIF1Î³; alias, TRIM33/RFG7/PTC7/ectodermin) belongs to an evolutionarily conserved family of nuclear factors that have been implicated in stem cell pluripotency, embryonic development, and tumor suppression. TIF1Î³ expression is markedly down-regulated in human pancreatic tumors, and Pdx1-driven Tif1Î³ inactivation cooperates with the Kras(G12D) oncogene in the mouse pancreas to induce intraductal papillary mucinous neoplasms. In this study, we report that aged Pdx1-Cre; LSL-Kras(G12D); Tif1Î³(lox/lox) mice develop pancreatic ductal adenocarcinomas (PDACs), an aggressive and always fatal neoplasm, demonstrating a Tif1Î³ tumor-suppressive function in the development of pancreatic carcinogenesis. Deletion of SMAD4/DPC4 (deleted in pancreatic carcinoma locus 4) occurs in approximately 50% of human cases of PDAC. We, therefore, assessed the genetic relationship between Tif1Î³ and Smad4 signaling in pancreatic tumors and found that Pdx1-Cre; LSL-Kras(G12D); Smad4(lox/lox); Tif1Î³(lox/lox) (alias, KSSTT) mutant mice exhibit accelerated tumor progression. Consequently, Tif1Î³ tumor-suppressor effects during progression from a premalignant to a malignant state in our mouse model of pancreatic cancer are independent of Smad4. These findings establish, for the first time to our knowledge, that Tif1Î³ and Smad4 both regulate an intraductal papillary mucinous neoplasm-to-PDAC sequence through distinct tumor-suppressor programs.

Tif1Î³ suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway.

NUPR1 (nuclear protein 1), also called P8 (molecular mass 8 kDa) or COM1 (candidate of metastasis 1), is involved in the stress response and in cancer progression. In the present study, we investigated whether human NUPR1 expression was regulated by TGFÎ² (transforming growth factor Î²), a secreted polypeptide largely involved in tumorigenesis. We demonstrate that the expression of NUPR1 was activated by TGFÎ² at the transcriptional level. We show that this activation is mediated by the SMAD proteins, which are transcription factors specifically involved in the signalling of TGFÎ² superfamily members. NUPR1 promoter analysis reveals the presence of a functional TGFÎ²-response element binding the SMAD proteins located in the genomic DNA region corresponding to the 5'-UTR (5'-untranslated region). Altogether, the molecular results of the present study, which demonstrate the existence of a TGFÎ²/SMAD/NUPR1 activation cascade, open the way to consider and investigate further a new mechanism enabling TGFÎ² to promote tumorigenesis by inducing stress resistance.

Article	Year
Transcription activation of FLRG and follistatin by activin A, through Smad proteins, participates in a negative feedback loop to modulate activin A function. Oncogene\| PubMed ID: 11948405	2002
Regulation of human erythropoiesis by activin A, BMP2, and BMP4, members of the TGFbeta family. Experimental cell research\| PubMed ID: 12531697	2003
FLRG, a new ADAM12-associated protein, modulates osteoclast differentiation. Biology of the cell / under the auspices of the European Cell Biology Organization\| PubMed ID: 15574124	2005
A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin. Experimental cell research\| PubMed ID: 16336961	2006
Identification of NF-kappaB responsive elements in follistatin related gene (FLRG) promoter. Gene\| PubMed ID: 17395406	2007
AF10-dependent transcription is enhanced by its interaction with FLRG. Biology of the cell / under the auspices of the European Cell Biology Organization\| PubMed ID: 17868029	2007
Generation of mice with conditionally activated transforming growth factor beta signaling through the TbetaRI/ALK5 receptor. Genesis (New York, N.Y. : 2000)\| PubMed ID: 18821589	2008
iNKT cell development is orchestrated by different branches of TGF-beta signaling. The Journal of experimental medicine\| PubMed ID: 19451264	2009
Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas. PLoS genetics\| PubMed ID: 19629168	2009
Tif1Î³ suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway. The American journal of pathology\| PubMed ID: 22469842	2012
The human NUPR1/P8 gene is transcriptionally activated by transforming growth factor Î² via the SMAD signalling pathway. The Biochemical journal\| PubMed ID: 22738338	2012

Sylvie Martel

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