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

Activin A, BMP2, and BMP4, 3 members of the transforming growth factor-beta family, are involved in the regulation of hematopoiesis. Here, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay. Our results highlight for the first time that, in the absence of thrombopoietin, BMP4 is able to induce CD34(+) progenitor differentiation into megakaryocytes through all stages. Although we have previously shown that activin A and BMP2 are involved in erythropoietic commitment, these molecules have no effect on human megakaryopoietic engagement and differentiation. Using signaling pathway-specific inhibitors, we show that BMP4, like thrombopoietin, exerts its effects on human megakaryopoiesis through the JAK/STAT and mTor pathways. Inhibition of the BMP signaling pathway with blocking antibodies, natural soluble inhibitors (FLRG or follistatin), or soluble BMP receptors reveals that thrombopoietin uses the BMP4 pathway to induce megakaryopoiesis, whereas the inverse is not occurring. Finally, we show that thrombopoietin up-regulates the BMP4 autocrine loop in megakaryocytic progenitors by inducing their production of BMP4 and up-regulating BMP receptor expression. In summary, this work indicates that BMP4 plays an important role in the control of human megakaryopoiesis.

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin 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.

We have previously generated a transgenic mouse strain (LSL-TÎ²RI(CA)) containing a Cre-inducible constitutively active TGFÎ² type I receptor (Bartholin et al., 2008, Genesis 46: 724-731). Transgene expression depends on the excision of a floxed-transcriptional STOP (LSL, Lox-STOP-Lox) located upstream the TÎ²RI(CA) coding sequence. To evaluate the correct excision of the STOP signal in the presence of Cre-recombinase, we developed a rapid screening based on an original PCR genotyping strategy. More precisely, we designed a set of primers flanking the LSL containing region. The size of the amplified products will differ according to recombination status of the LSL-TÎ²RI(CA) allele. Indeed, the size of the STOP containing PCR product is 1.93 kb, but is reduced to 0.35 kb when the STOP signal is removed after Cre-mediated recombination. We validated excision in several compartments, including pancreas, liver, T lymphocytes, and embryos using different Cre expressing transgenic mouse strains. This represents a simple and efficient way of monitoring the tissue specific recombination of the LSL-TÎ²RI(CA) allele.

A rapid strategy to detect the recombined allele in LSL-TÎ²RICA transgenic mice.

Lysyl Oxidase (LOX) is an extracellular enzyme involved in the maturation of connective tissues. It also acts in many cell types as a regulator of cell behaviour and phenotype through intracellular signalling pathways. Recently, LOX was shown to be present in human epidermis where its precise functions remain unclear. We showed here that in confluent monolayer cultures of normal human keratinocytes (KCs) and N/TERT-1-immortalized KCs, LOX expression was induced during the first differentiation steps. Moreover, the silencing of LOX by stable RNA interference disrupted the expression of early differentiation markers. In a reconstructed-epidermis model, LOX silencing did not impair the stratification process nor the formation of the first differentiated layers. However, terminal differentiation was strongly impaired, as shown by a decreased expression of late differentiation proteins and by the absence of stratum corneum. Nonetheless, inhibition of LOX enzymatic activity by Î²-aminopropionitrile did not affect the differentiation process. Therefore, LOX protein acts during the first steps of KC differentiation and is important for subsequent commitment into terminal differentiation. Taken together, these results suggest that a finely regulated expression of LOX is necessary for normal KC differentiation and thus for maintenance of epidermal homeostasis.

Lysyl oxidase silencing impairs keratinocyte differentiation in a reconstructed-epidermis model.

Adaptation to hypoxia is a driving force for tumor progression that leads to therapy resistance and poor clinical outcome. Hypoxic responses are mainly mediated by hypoxia-inducible transcription factor-1 (HIF-1). One critical HIF-1 target mediating tumor progression is lysyl oxidase (LOX), which catalyzes cross-linking of collagens and elastin in the extracellular matrix, thereby regulating tissue tensile strength. Paradoxically, LOX has been reported to be both upregulated and downregulated in cancer cells, especially in colorectal cancer. Thus, we hypothesized that LOX might regulate expression of HIF-1 to create a self-timing regulatory circuit. Using human colorectal carcinoma cell lines in which HIF-1 and LOX expression could be modulated, we showed that LOX induction enhanced HIF-1 expression, whereas LOX silencing reduced it. Mechanistic investigations revealed that LOX activated the PI3K (phosphoinositide 3-kinase)-Akt signaling pathway, thereby upregulating HIF-1Î± protein synthesis in a manner requiring LOX-mediated hydrogen peroxide production. Consistent with these results, cancer cell proliferation was stimulated by secreted and active LOX in an HIF-1Î±-dependent fashion. Furthermore, nude mice xenograft assays established that HIF-1 potentiated LOX action on tumor growth in vivo. Taken together, these findings provide compelling evidence that LOX and HIF-1 act in synergy to foster tumor formation, and they suggest that HIF-1/LOX mutual regulation is a pivotal mechanism in the adaptation of tumor cells to hypoxia.

The HIF-1-inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth.

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.

Bastien Kaniewski

INSERM U1052,
CNRS UMR5286

Centre de Recherche en Cancérologie de Lyon

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Bastien Kaniewski

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Centre de Recherche en Cancérologie de Lyon

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

INSERM U1052,
CNRS UMR5286