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NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells and suppresses PDAC initiation

Isidoro Cobo, Sumit Paliwal, Júlia Melià-Alomà1, Ariadna Torres1, Jaime Martínez-Villarreal, Fernando García, Irene Millán, Natalia del Pozo, Joo-Cheol Park2, Raymond J. MacDonald3, Javier Munoz, Francisco X. Real1 •
Pompeu Fabra University1, Seoul National University2, University of Texas Southwestern Medical Center3
09 Aug 2021-bioRxiv (Cold Spring Harbor Laboratory)-
TL;DR: In this paper, NFIC binding sites are found at very short distances from NR5A2-bound genomic regions and both proteins co-occur in the same complex and NFIC dampens the ER stress program through its binding to ER stress gene promoters and is required for complete resolution of Tunicamycin-mediated ER stress.
Abstract: Tissue-specific differentiation is driven by specialized transcriptional networks. Pancreatic acinar cells crucially rely on the PTF1 complex, and on additional transcription factors, to deploy their transcriptional program. Here, we identify NFIC as a novel regulator of acinar differentiation using a variety of methodological strategies. NFIC binding sites are found at very short distances from NR5A2-bound genomic regions and both proteins co-occur in the same complex. Nfic knockout mice show reduced expression of acinar genes and, in ChIP-seq experiments, NFIC binds the promoters of acinar genes. In addition, NFIC binds to the promoter of, and regulates, genes involved in RNA and protein metabolism; in Nfic knockout mice, p-RS6K1 and p-IEF4E are down-regulated indicating reduced activity of the mTOR pathway. In 266-6 acinar cells, NFIC dampens the ER stress program through its binding to ER stress gene promoters and is required for complete resolution of Tunicamycin-mediated ER stress. Normal human pancreata from subjects with low NFIC mRNA levels display reduced epxression of genes down-regulated in Nfic knockout mice. Consistently, NFIC displays reduced expression upon induced acute pancreatitis and is required for proper recovery after damage. Finally, expression of NFIC is lower in samples of mouse and human pancreatic ductal adenocarcinoma and Nfic knockout mice develop an increased number of mutant Kras-driven pre-neoplastic lesions.

Summary (3 min read)

Jump to: [INTRODUCTION] – [Identification of novel transcription factors involved in the regulation of pancreatic] – [DISCUSSION] – [NFIC regulates expression of ribosomal genes and mitigates ER stress in the] – [NFIC is dynamically regulated during pancreatitis and cancer.] – [MATERIAL AND METHODS] – [Histology, immunofluorescence (IF) and immunohistochemical (IHC) analyses.] – [Quantitative RT-PCR (RT-qPCR).] – [Chromatin immunoprecipitation (ChIP).] and [Gene Set Enrichment Analysis (GSEA).]

INTRODUCTION

  • Pancreatic acinar cells are highly specialized protein synthesis factories that have a well-developed rough endoplasmic reticulum (ER), a prominent Golgi complex, and abundant secretory granules 1 .
  • Monoallelic or homozygous inactivation of several acinar transcriptional regulators in the germline, the embryonic pancreas, or the adult pancreas can result in compromised acinar function that favors loss of cellular identity and poises acinar cells for transformation upon activation of mutant KRas 10, 11, 12 .
  • Additional roles have been proposed through the regulation of Trp53 15, 16, 17 .
  • (which was not certified by peer review) is the author/funder.
  • Using a combination of omics analyses and studies in knockout mice and cultured cells, the authors now uncover novel roles of NFIC as a regulator of acinar function whose major impact is at the level of the ER stress response in murine and human pancreas.

Identification of novel transcription factors involved in the regulation of pancreatic

  • To discover novel transcription factors that might cooperate with known acinar regulators (e.g. PTF1A, GATA6, NR5A2, and MIST1), the authors reanalyzed publicly available ChIP-sequencing data and used HOMER to search for motifs enriched in the sequencing reads.
  • Motifs corresponding to RBPJ/RBPJL and HNF1, known regulators of acinar differentiation, were also enriched, thus validating the strategy applied.
  • Of all NFI family members, NFIC is expressed at highest levels in both mouse and human pancreas ; therefore, the authors focused on NFIC for further study.
  • Analysis of the published ChIP-Seq data revealed several PTF1A and NR5A2 peaks in the proximal Nfic promoter and the binding was confirmed by ChIP-qPCR , strongly suggesting that Nfic is a PTF1A and NR5A2 target.
  • The copyright holder for this preprint this version posted August 9, 2021.

DISCUSSION

  • Increasing evidence supports an "analog" differentiation model whereby additional TFs are required for "completion" of this process.
  • The conservation of spacing between NR5A2 and NFIC motifs among NR5A2 target genes supports transcriptional cooperation.
  • Comparison of NR5A2 ChIP-Seq data from embryonic and adult pancreas indicates that the role of NFIC is mainly in the latter, supporting its requirement for completion of acinar maturation and highlighting a functional role distinct from that of NR5A2.
  • NFI proteins were first proposed to be involved in the regulation of ubiquitous genes but they can also regulate tissue-specific genes 13 , including CEL in the mammary gland and DSPP in odontoblasts 14, 19 .
  • These findings suggest that the activation of pro-inflammatory phenotypes in mice in which acinar cells fail to acquire normal maturation can result from both direct (NR5A2 and GATA6) 4, 7, 10, 12 and indirect (PTF1A and NFIC) mechanisms, in agreement with the ChIP-Seq data available.

NFIC regulates expression of ribosomal genes and mitigates ER stress in the

  • Accordingly, a coordinated down-regulation of gene sets related to the digestive process and to protein metabolism and oxidative phosphorylation occurs in adult Nfic -/pancreata.
  • The mTOR pathway is a central actor in protein biosynthesis and autophagy and, therefore, a candidate mediator of this phenotype (reviewed in [33]).
  • The authors found reduced levels of P-RS6K1, its substrate P-RS6, and P-EIF4E, together with an up-regulation of P-ERK, in acinar cells of Nfic -/mice.
  • The finding that NFIC -but not NR5A2 -binds the promoter of ER stress genes suggests a distinct role of the former in this process.
  • A re-analysis of published data shows that Nfic is down-regulated in Nr5a2 -/hepatocytes in basal conditions (fold change of -0.54) 35 , suggesting that the deficient response to TM in Nr5a2 -/hepatocytes might partially occur through Nfic down-regulation.

NFIC is dynamically regulated during pancreatitis and cancer.

  • A failure to achieve complete acinar maturation is associated with more severe damage and delayed recovery during caerulein-mediated pancreatitis, as shown upon inactivation of Gata6, Mist1, and Ptf1a in the pancreas 4, 44 .
  • The role of NFIC in acinar cell differentiation and mitigation of ER stress suggested a contribution during tumorigenesis.
  • The copyright holder for this preprint this version posted August 9, 2021.
  • TFs previously described, the role of NFIC is restricted to the adult pancreas and distinctly affects RNA and protein metabolism and the UPR-mediated ER stress.

MATERIAL AND METHODS

  • All crosses were maintained in a predominant C57BL/6 background.
  • In brief, animals were weighed before the procedure and caerulein was administered intraperitoneally.
  • For the glucose tolerance test, male mice were fasted for 16 h and basal glycaemia was measured in tail blood.
  • For most experiments, >5 mice per group were used.

Histology, immunofluorescence (IF) and immunohistochemical (IHC) analyses.

  • Histological processing was performed using standard procedures.
  • After washing with PBS, sections were mounted with Prolong Gold Antifade Reagent (Life Technology).
  • Sections were incubated with 2% BSA-PBS for 1h at room temperature, and then with the primary antibody overnight at 4 ºC.
  • Briefly, areas of interest (AOI) were selected for quantification and then exported as individual TIFF images.
  • For quantification of KI67 + positive cells, at least 10 random images from each pancreas were selected and only positive acinar cells were quantified.

Quantitative RT-PCR (RT-qPCR).

  • Total RNA was treated with DNase I for 30 min at 37 °C and cDNAs were prepared according to the manufacturer's specifications, using the TaqMan reverse transcription reagents (Applied (which was not certified by peer review) is the author/funder.
  • For immunoprecipitation of proteins from fresh total pancreas lysates, a piece of mouse pancreas was isolated and minced in 50 mM Tris-HCl pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40 containing 3× phosphatase inhibitor cocktail (Sigma-Aldrich) and 3× EDTA-free complete protease inhibitor cocktail .
  • Afterwards, beads were washed three times with coupling buffer and once with 1 M Tris pH 9.
  • Protein lysates (10-15 mg, tissues) were then incubated overnight at 4 °C with antibody-coated dynabeads (Thermo Fisher Scientific).

Chromatin immunoprecipitation (ChIP).

  • Pancreas tissue was minced, washed with cold PBS supplemented with 3× protease and phosphatase cocktail inhibitors, and then fixed with 1% formaldehyde for 20 min at room temperature.
  • The supernatant was collected after centrifugation and chromatin was sonicated with a Covaris instrument for 40 min (20% duty cycle; 10% intensity; 200 cycle), yielding DNA fragments with a bulk size of 300-500 bp.
  • Data were analysed using the nextpresso pipeline http://bioinfo.cnio.es/nextpresso/).

Gene Set Enrichment Analysis (GSEA).

  • A ranking metric [-log10(p value)/sign(log2FoldChange)] was used to generate a ranked gene list from the DEseq output.
  • The resulting libraries were sequenced on Illumina HiSeq 2500, v4 Chemistry.
  • The copyright holder for this preprint this version posted August 9, 2021.
  • Among the two replicates, the one with highest number of identified target genes was taken: replicate 1 of NFIC ChIP-Seq in GM12878, NFIC ChIP-Seq in HepG2 and NFIC ChIP-Seq in SK-N-SH and replicate 2 of NFIC ChIP-Seq in ECC1 cell line.

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Figures (1)

Content maybe subject to copyright    Report

NFIC regulates ribosomal biology and ER stress in pancreatic acinar cells
1
and suppresses PDAC initiation
2
3
4
Isidoro Cobo,
1,2
Sumit Paliwal,
1
Júlia Melià-Alomà,
1,3
Ariadna Torres,
1,3
5
Jaime Martínez-Villarreal,
1
Fernando García,
4
Irene Millán,
1,2
Natalia del Pozo,
1,2
Joo-
6
Cheol Park,
5
Ray J. MacDonald,
6
Javier Muñoz,
4
and Francisco X. Real
1-3
7
8
9
Short title: NFIC in pancreatic homeostasis and cancer
10
11
1
Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre-CNIO,
12
Madrid, Spain
13
2
CIBERONC, Madrid, Spain
14
3
Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra,
15
Barcelona, Spain
16
4
Proteomics Unit, Spanish National Cancer Research Centre-CNIO, Madrid, Spain.
17
ProteoRed - ISCIII
18
5
Department of Oral Histology-Developmental Biology, School of Dentistry, Seoul
19
National University, Seoul, Korea
20
6
Department of Molecular Biology, University of Texas Southwestern Medical Center,
21
Dallas, TX, USA
22
23
24
25
Correspondence: Francisco X. Real
26
Centro Nacional de Investigaciones Oncológicas-CNIO
27
Melchor Fernández Almagro, 3
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28029-Madrid, Spain
29
E-mail: preal@cnio.es
30
31
32
Conflicts of interest: none to declare
33
34
35
Funding: This work was supported, in part, by grants SAF2011-29530, SAF2015-70553-
36
R, and RTI2018-101071-B-I00 from Ministerio de Ciencia, Innovación y Universidades
37
(Madrid, Spain) (co-funded by the ERDF-EU) and RTICC from Instituto de Salud Carlos
38
III (RD12/0036/0034) to FXR. IC was recipient of a Beca de Formación del Personal
39
Investigador from Ministerio de Economía y Competitividad (Madrid, Spain). The
40
research leading to these results has received funding from People Programme (Marie
41
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
2
Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-
42
2013) (REA grant agreement n° 608765”). SP was supported by a Juan de la Cierva
43
Programme fellowship from Ministerio de Ciencia, Innovación y Universidades. IM was
44
supported by a Fellowship from Fundació Bancaria La Caixa (ID 100010434) (grant
45
number LCF/BQ/ES18/11670009). CNIO is supported by Ministerio de Ciencia,
46
Innovación y Universidades as a Centro de Excelencia Severo Ochoa SEV-2015-0510.
47
48
Statement of author contributions
49
IC: study concept and design; acquisition of data; analysis and interpretation of data;
50
statistical analysis; drafting of the manuscript;
51
SP: acquisition of data; analysis and interpretation of data; drafting of the manuscript
52
JMA: acquisition of data; analysis and interpretation of data; drafting of the manuscript
53
AT: acquisition of data; analysis and interpretation of data;
54
JMV: analysis and interpretation of data;
55
FG: acquisition of data; analysis and interpretation of data;
56
IM: analysis and interpretation of data;
57
NdP: technical support and acquisition of data;
58
JCP: material support;
59
RJM: critical revision of the data and important intellectual content;
60
JM: acquisition of data; analysis and interpretation of data;
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FXR: study concept and design; analysis and interpretation of data; drafting of the
62
manuscript; overall study supervision; obtained funding.
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All authors provided input about manuscript content.
64
Accession numbers: RNA sequencing data have been deposited in GEO with
65
accession number GSE126907 and NFIC ChIP sequencing data have been deposited
66
in GEO with accession number GSE181098
67
68
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
3
ABSTRACT
69
70
Tissue-specific differentiation is driven by specialized transcriptional networks.
71
Pancreatic acinar cells crucially rely on the PTF1 complex, and on additional
72
transcription factors, to deploy their transcriptional program. Here, we identify NFIC as a
73
novel regulator of acinar differentiation using a variety of methodological strategies. NFIC
74
binding sites are found at very short distances from NR5A2-bound genomic regions and
75
both proteins co-occur in the same complex. Nfic knockout mice show reduced
76
expression of acinar genes and, in ChIP-seq experiments, NFIC binds the promoters of
77
acinar genes. In addition, NFIC binds to the promoter of, and regulates, genes involved
78
in RNA and protein metabolism; in Nfic knockout mice, p-RS6K1 and p-IEF4E are down-
79
regulated indicating reduced activity of the mTOR pathway. In 266-6 acinar cells, NFIC
80
dampens the ER stress program through its binding to ER stress gene promoters and is
81
required for complete resolution of Tunicamycin-mediated ER stress. Normal human
82
pancreata from subjects with low NFIC mRNA levels display reduced epxression of
83
genes down-regulated in Nfic knockout mice. Consistently, NFIC displays reduced
84
expression upon induced acute pancreatitis and is required for proper recovery after
85
damage. Finally, expression of NFIC is lower in samples of mouse and human pancreatic
86
ductal adenocarcinoma and Nfic knockout mice develop an increased number of mutant
87
Kras-driven pre-neoplastic lesions.
88
89
Word count: 211
90
91
Keywords: NFIC, pancreas, acinar differentiation, ribosome, endoplasmic reticulum
92
stress, unfolded protein response, transcriptional networks, pancreatitis, pancreatic
93
cancer
94
95
Abbreviations: ChIP, chromatin immunoprecipitation; DEG, differentially expressed
96
genes; EMT, epithelial-mesenchymal transition; ER, endoplasmic reticulum; GSEA,
97
Gene set enrichment analysis; IF, immunofluorescence; IHC; immunohistochemistry;
98
PDAC, pancreatic ductal adenocarcinoma; TF, transcription factor; TM, tunicamycin;
99
UPR, unfolded protein response.
100
101
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
4
INTRODUCTION
102
103
Pancreatic acinar cells are highly specialized protein synthesis factories that
104
have a well-developed rough endoplasmic reticulum (ER), a prominent Golgi complex,
105
and abundant secretory granules
1
. Acinar differentiation is contingent on the activity of a
106
master regulator, the adult PTF1 complex, composed of the pancreas-specific
107
transcription factors (TFs) PTF1A and RPBJL and the ubiquitous protein E47
2,3
. PTF1
108
binds the proximal promoter of genes coding for digestive enzymes, secretory proteins
109
and other TFs, and activates their expression. The PTF1 complex is the main driver of
110
acinar differentiation but additional TF with tissue-restricted expression patterns are
111
implicated in the fine-tuning of this process, including GATA6
4
, MIST1
5
, and
112
NR5A2/LRH-1
6,7
. Acinar cells play a crucial role in acute and chronic pancreatitis, two
113
common and disabling conditions. Recent work using genetic mouse models has shown
114
that, upon expression of mutant KRas, acinar cells can be the precursors of Pancreatic
115
Intraepithelial Neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC)
8,9
.
116
Our laboratory and others have shown that the acinar differentiation program acts
117
as a tumor suppressor in the pancreas. Monoallelic or homozygous inactivation of
118
several acinar transcriptional regulators in the germline, the embryonic pancreas, or the
119
adult pancreas can result in compromised acinar function that favors loss of cellular
120
identity and poises acinar cells for transformation upon activation of mutant KRas
10,11,12
.
121
The tumor suppressive function of these TF is not obvious because the exocrine
122
pancreas has a large functional reserve, i.e. massive alterations in cellular function need
123
to occur in order to be reflected in histological or clinical changes.
124
Here, we use bioinformatics tools to identify NFIC as a novel acinar regulator.
125
NFIC is a member of the nuclear factor I family of TFs that regulate both ubiquitous and
126
tissue-restricted genes
13
. In the mammary gland, NFIC activates the expression of milk
127
genes involved in lactation
14
. Furthermore, it acts as a breast cancer tumor suppressor,
128
as it directly represses the expression of Ccnd1 and Foxf1, a potent inducer of epithelial-
129
mesenchymal transition (EMT), invasiveness, and tumorigenicity. Additional roles have
130
been proposed through the regulation of Trp53
15,16,17
. The physiological role of NFIC has
131
been best studied in dentinogenesis, since Nfic
-/-
mice develop short molar roots and
132
display aberrant odontoblast differentiation and dentin formation
18
. NFIC regulates
133
odontoblast-related genes, including Dssp
19
, Wnt
20
, and hedgehog signaling
21
.
134
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
5
Using a combination of omics analyses and studies in knockout mice and cultured
135
cells, we now uncover novel roles of NFIC as a regulator of acinar function whose major
136
impact is at the level of the ER stress response in murine and human pancreas. Unlike
137
most other TFs previously identified as required for full acinar function, NFIC belongs to
138
a novel family of acinar regulators with tissue-wide expression. NFIC dysregulation
139
sensitizes the pancreas to damage and neoplastic transformation.
140
141
142
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprintthis version posted August 9, 2021. ; https://doi.org/10.1101/2021.08.09.455477doi: bioRxiv preprint
Citations
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Journal Article•DOI•
Gene expression signatures of individual ductal carcinoma in situ lesions identify processes and biomarkers associated with progression towards invasive ductal carcinoma

[...]

Clare A. Rebbeck, Jian Xian, Susanne Bornelöv, Joseph Geradts, Amy Hobeika, Heather Geiger, Jose Franco Alvarez, Elena Rozhkova, Ashley Nicholls, Nicolas Robine, H. Kim Lyerly, Gregory J. Hannon 
13 Jun 2022-Nature Communications
TL;DR: In this article , gene expression analysis from 2,000 individually micro-dissected ductal lesions representing 145 patients was performed to identify early processes and potential biomarkers, including CAMK2N1 , MNX1 , ADCY5 , HOXC11 and ANKRD22 , whose reduced expression is associated with the progression of DCIS to invasive breast cancer.
Abstract: Abstract Ductal carcinoma in situ (DCIS) is considered a non-invasive precursor to breast cancer, and although associated with an increased risk of developing invasive disease, many women with DCIS will never progress beyond their in situ diagnosis. The path from normal duct to invasive ductal carcinoma (IDC) is not well understood, and efforts to do so are hampered by the substantial heterogeneity that exists between patients, and even within patients. Here we show gene expression analysis from > 2,000 individually micro-dissected ductal lesions representing 145 patients. Combining all samples into one continuous trajectory we show there is a progressive loss in basal layer integrity heading towards IDC, coupled with two epithelial to mesenchymal transitions, one early and a second coinciding with the convergence of DCIS and IDC expression profiles. We identify early processes and potential biomarkers, including CAMK2N1 , MNX1 , ADCY5 , HOXC11 and ANKRD22 , whose reduced expression is associated with the progression of DCIS to invasive breast cancer.

10 citations

Journal Article•DOI•
Transcriptome analysis of the response to thyroid hormone in Xenopus neural stem and progenitor cells

[...]

Camila Cordero-Véliz, Juan Larraín, Fernando Faunes
06 Sep 2022-Developmental Dynamics
TL;DR: This work provides new insights into the dynamics of activation of NPSCs in vivo in response to T3 during a critical period of neurogenesis in the metamorphosis, suggesting that NSPCs activation induced by T3 is characterized by an early proteome remodeling through the synthesis of the translation machinery and the degradation of proteins by the proteasome.
Abstract: The thyroid hormones—thyroxine (T4) and 3,5,3′triiodothyronine (T3)—regulate the development of the central nervous system (CNS) in vertebrates by acting in different cell types. Although several T3 target genes have been identified in the brain, the changes in the transcriptome in response to T3 specifically in neural stem and progenitor cells (NSPCs) during the early steps of NSPCs activation and neurogenesis have not been studied in vivo. Here, we characterized the transcriptome of FACS‐sorted NSPCs in response to T3 during Xenopus laevis metamorphosis.

2 citations

Posted Content•DOI•
Creating a ‘Timeline’ of ductal carcinoma in situ to identify processes and biomarkers for progression towards invasive ductal carcinoma

[...]

Clare A. Rebbeck, Jian Xian, Susanne Bornelöv, Joseph Geradts, Amy Hobeika, Heather Geiger, Jose Franco Alvarez, Elena Rozhkova, Ashley Nicholls, Nicolas Robine, H. Kim Lyerly, Gregory J. Hannon 
04 Mar 2022-bioRxiv
TL;DR: A ‘Timeline’ of disease progression is generated, utilising the variability within patients and combining >2,000 individually micro-dissected ductal lesions from 145 patients into one continuous trajectory, showing there is a progressive loss in basal layer integrity, coupled with two epithelial to mesenchymal transitions (EMT) early in the timeline.
Abstract: Ductal carcinoma in situ (DCIS) is considered a non-invasive precursor to breast cancer, and although associated with an increased risk of developing invasive disease, many women with DCIS will never progress beyond their in situ diagnosis. The path from normal duct to invasive disease is not well understood, and efforts to do so are hampered by the substantial heterogeneity that exists between patients and even within patients. Using gene expression analysis, we have generated a ‘Timeline’ of disease progression, utilising the variability within patients and combining >2,000 individually micro-dissected ductal lesions from 145 patients into one continuous trajectory. Using this Timeline we show there is a progressive loss in basal layer integrity, coupled with two epithelial to mesenchymal transitions (EMT), one early in the timeline and a second just prior to cells leaving the duct. We identify early processes and potential biomarkers, including CAMK2N1, MNX1, ADCY5, HOXC11 and ANKRD22, whose reduced expression is associated with the progression of DCIS to invasive breast cancer.
References
More filters
Journal Article•DOI•
Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells

[...]

R. Maynard Case1•
University of Sydney1
01 May 1978-Biological Reviews
TL;DR: This review traces the pathway of amino acids as they are taken up by the acinar cell, incorporated into digestive hydrolases, transported through the cell and finally discharged from the cell, and considers the mechanisms by which these steps are controlled.
Abstract: SUMMARY 1After an outline description of pancreatic structure and function, and a more detailed account of acinar cell morphology, this review traces the pathway of amino acids as they are taken up by the acinar cell, incorporated into digestive hydrolases, transported through the cell and finally discharged from the cell, and considers the mechanisms by which these steps are controlled. At all stages comparisons are made with other secretory cells. 2The use of radioautography and cell-fractionation techniques in determining this pathway in the pancreas are described. The route and kinetics of the process in pancreas are compared with those in other cells. 3Amino-acid entry is by an active mechanism. However the intracellular pool of accumulated amino acids may not be used directly in protein synthesis. Selection of amino acids for incorporation into proteins may occur whilst they are associated with carrier systems within the plasma membrane. There is no convincing evidence that amino-acid entry can be influenced by the pancreatic secretagogues, cholecystokin-pancreazymin (CCK-PZ) or acetylcholine. 4Secretory proteins are synthesized on ribosomes bound to the endoplasmic reticulum (ER) and the nascent proteins vectorially transferred across the ER membrane into the ER cisternae. All messenger RNA molecules which are templates for secretory proteins appear to possess an initial sequence of codons whose translation produces a ‘signal’ sequence of amino acids. This signal sequence somehow triggers attachment of the ribosomes to the ER, thereby automatically determining that the final translation product is destined for the ER cisternae. 5The effects of CCK-PZ and acetylcholine on pancreatic protein synthesis are controversial. Whereas stimulation can be observed in vivo, this has not been convincingly demonstrated in vitro. I conclude that while CCK-PZ and acetylcholine may accelerate protein synthesis, the physiological significance of this effect remains to be clarified. Long-term stimulation can modify pancreatic enzyme synthesis and this, together with other factors, may be the means of dietary adaptation by the gland. 6Newly synthesized proteins travel from the ER cisternae via the peripheral Golgi components to the Golgi cisternae. Transport from ER to Golgi cisternae may occur by a vesicle shuttle service or by direct tubular connexions. Although sustained stimulation with CCK-PZ analogues can accelerate this intracellular transport step, pancreatic secretagogues have not yet been shown to accelerate transport under physiological conditions. 7The Golgi complex has a number of functions including: glycosylation and, where appropriate, sulphation of glycoprotein and mucopolysaccharide components of the zymogen granules (ZG) and granule membranes; sequestration of divalent cations which bind to secretory proteins; the formation of condensing vacuoles (CV) from the inner Golgi cisternae. 8Aggregation of proteins occurs passively within CV so as to form osmotically inert complexes, thereby reducing internal osmotic activity and causing water to diffuse out. This condensation imparts a gel-like consistency to the mature ZG so formed. 9Discharge of ZG occurs by a process of exocytosis involving fusion of the ZG membrane with the apical plasma membrane, release of the ZG contents, and retrieval of the ZG membrane from the plasma membrane by endocytotic mechanisms. The mechanisms responsible for migration of ZG towards the cell apex and for exocytosis remain unknown but may involve the participation of microtubules and/or microfilaments. Although there is a small, basal discharge of ZG at all times, stimulation with CCK-PZ or acetylcholine greatly accelerates the process. 10The basic tenet of the secretory mechanism summarized above is that, following synthesis, secretory proteins are confined within an intracellular organelle at all times. This ‘segregation’ hypothesis has been challenged by the ‘equilibrium’ hypothesis in which secretory proteins are suggested to move across cellular membranes and are therefore at equilibrium within the various compartments of the cell. While many of the observations on which the equilibrium hypothesis are based are tenuous, some others cannot readily be explained by the segregation model. Proponents of the equilibrium hypothesis therefore suggest that preferential release of individual hydrolases from ZG occurs, followed by their separate transport across the apical cell membrane. The claims of this alternative model are discussed. 11In the final section are discussed the intracellular mechanisms by which CCK-PZ and acetylcholine act on the acinar cell to cause discharge. The overall membrane perturbations brought about by CCK-PZ and acetylcholine appear to be the same and include cell depolarization, and perhaps increased phospholipid turnover. Both events may be related to an altered membrane permeability to cations. CCK-PZ, but not acetylcholine, will activate adenylate cyclase, but cyclic AMP does not appear to be involved in regulating enzyme discharge. Instead, Ca2+ is the major intracellular second messenger. However, rather than increase Ca2+ uptake into the cell, CCK-PZ and acetylcholine appear to raise the intracellular Ca2+ concentration by causing release of Ca2+ from intracellular stores. The mechanism by which they do this, and the role of Ca2+ in the discharge process remain unknown.

267 citations

Journal Article•DOI•
The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity.

[...]

Christopher L. Pin1, J. Michael Rukstalis2, Charis L. Johnson1, Stephen F. Konieczny2•
University of Western Ontario1, Purdue University2
12 Nov 2001-Journal of Cell Biology
TL;DR: It is proposed that Mist1KO mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity.
Abstract: The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, we have examined Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells. Mist1-null (Mist1KO) mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1KO mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1KO mice. Based on these observations, we propose that Mist1KO mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity.

253 citations

Journal Article•DOI•
PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L.

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Thomas M. Beres1, Thomas M. Beres2, Toshihiko Masui2, Galvin H. Swift, Ling Shi2, Ling Shi3, R. Michael Henke2, Raymond J. MacDonald •
University of Utah1, University of Texas Southwestern Medical Center2, University of Toronto3
01 Jan 2006-Molecular and Cellular Biology
TL;DR: The presence of related peptide motifs in other transcription factors indicates a broader Notch-independent function for RBPJ/SU(H), which indicates that the association of P48 and RBPs is required for proper embryonic development.
Abstract: One of the most intriguing properties of biological regulatory schemes is the certainty of evolutionary variations from an original theme. The definition of a canonical scheme nearly guarantees the discovery of an alternative in which a useful regulator is recruited for other purposes. In this regard, the canonical Notch-signaling pathway, which regulates cell fate decisions via a transcriptional off-on switch, is a useful example (2, 35). In the absence of signaling, a CSL-factor [CBF1/RBPJ/RBPSUH in mammals; Su(H) in Drosophila melanogaster; LAG-1 in nematodes] binds and represses target promoters by recruiting a corepressor complex. Binding of any of the family of DSL cell-surface ligands (Delta, Serrate/Jagged, and Lag-2) to the Notch receptor triggers cleavage of Notch and the release of its intracellular domain (NotchIC). The NotchIC enters the nucleus, binds CSL, displaces the corepressor, and recruits coactivators. Recently, a variation has been described in which the transcriptional effect is mediated independently of the CSL (29). Conversely, CSL appears to play a Notch-independent role in at least one developmental context in Drosophila (3). We describe here a novel Notch-independent function of the mammalian CSL (hereafter RBP-J) and its paralogue, RBP-L, by recruitment into PTF1, a basic helix-loop-helix (bHLH) transcription factor complex that controls pancreas-specific gene transcription. The pancreas is a multifunctional gland composed of both endocrine and exocrine tissues. The exocrine tissue comprises more than 90% of the adult pancreas and is composed of acini, which secrete digestive enzymes, and ducts, which secrete fluid and transport the acinar enzymes to the duodenum. Massive synthesis of the digestive enzymes is reflected in the pancreatic mRNA population: nearly 90% of the mRNA from the entire gland encodes a small number (about 20) of acinar secretory enzymes, such as amylases, elastases, chymotrypsinogens, and carboxypeptidases (12). The selective transcription of the acinar specific genes at such a high level is controlled largely by the PTF1 complex (6, 32). However, the mechanism of target-gene activation by PTF1 is unknown. Functional binding sites for the PTF1 complex are present in the 5′ promoter regions of all of the acinar digestive enzyme genes examined (6, 31). The binding site from the elastase 1 gene (Ela1) provides a model for the interaction of PTF1 with DNA and acinar cell-specific transcriptional activation. This site, known as the A element of the Ela1 enhancer, is located about 100 bp upstream of the 5′ end of Ela1, is necessary and sufficient to direct acinar specific expression in transgenic mice (30), and in situ cooperates with two nearby elements (B and C) to direct the high level of transcriptional activation characteristic of Ela1 (18). PTF1 is an unusual heterotrimeric bHLH transcription factor composed of PTF1a/P48 (a pancreas and neural specific bHLH protein), one of the common class A bHLH proteins, and a previously unidentified subunit (32, 33). (For clarity, we retain the use of p48/P48 [gene/protein], rather than Ptf1a/PTF1A, to distinguish the P48 subunit from the PTF1 complex.) PTF1 binding sites are bipartite with an E-box (preferably CACCTG) and a TC-box (TTTCCCA) spaced one or two helical turns apart, center to center (6, 31). Targeted deletion of the p48 gene causes pancreatic and cerebellar agenesis (14, 17, 36), so understanding the mechanism of transcriptional activation by PTF1 in differentiated acini will likely give insights into PTF1 action during pancreas and brain development as well. We show that the previously unidentified third subunit of PTF1 from adult pancreas is RBP-L, an organ-specific mammalian variant of the CSL proteins. RBP-L provides the high activation potential of the complex, which is dependent on contact of all three subunits of the complex with DNA. A similar transcriptionally active complex can be reconstituted with RBP-J, the mediator of Notch signaling. The interaction of P48 with the RBP subunits requires two peptide motifs conserved in P48s from insects to mammals. One or both of these peptides are deleted in families with heritable permanent neonatal diabetes mellitus, in which infants are born without a pancreas and cerebellum (36). The similar developmental consequences for neonatal mice without P48 and infants with mutant P48 unable to bind RBP-J or -L suggest that most or all of the developmental functions of P48 require its ability to recruit an RBP into a PTF1 complex. Motifs similar to the RBP-interacting sites of P48 are present in other transcription factors; therefore, PTF1 may be one of a new family of complexes that use RBP-J in a Notch-independent manner.

203 citations

Journal Article•DOI•
Essential role for NFI-C/CTF transcription-replication factor in tooth root development.

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George Steele-Perkins1, Kenneth G. Butz1, Kenneth G. Butz2, Gary E. Lyons3, Margarita Zeichner-David4, Heung-Joong Kim, Moon-Il Cho, Richard M. Gronostajski1, Richard M. Gronostajski5, Richard M. Gronostajski2 •
Cleveland Clinic1, University at Buffalo2, University of Wisconsin-Madison3, University of Southern California4, Case Western Reserve University5
01 Feb 2003-Molecular and Cellular Biology
TL;DR: The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid the understanding of postnatal tooth development.
Abstract: The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and Nfix. In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic−/− mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic−/− mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic−/− animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.

193 citations

Journal Article•DOI•
Metabolism of pancreatic cancer: paving the way to better anticancer strategies.

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Cheng Qin1, Gang Yang1, Jinshou Yang1, Bo Ren1, Huanyu Wang1, Guangyu Chen1, Fangyu Zhao1, Lei You1, Weibin Wang1, Yupei Zhao1 •
Peking Union Medical College Hospital1
02 Mar 2020-Molecular Cancer
TL;DR: Investigations of metabolism not only benefit the understanding of carcinogenesis and cancer progression but also provide new insights for treatments against pancreatic cancer.
Abstract: Pancreatic cancer is currently one of the most lethal diseases. In recent years, increasing evidence has shown that reprogrammed metabolism may play a critical role in the carcinogenesis, progression, treatment and prognosis of pancreatic cancer. Affected by internal or external factors, pancreatic cancer cells adopt extensively distinct metabolic processes to meet their demand for growth. Rewired glucose, amino acid and lipid metabolism and metabolic crosstalk within the tumor microenvironment contribute to unlimited pancreatic tumor progression. In addition, the metabolic reprogramming involved in pancreatic cancer resistance is also closely related to chemotherapy, radiotherapy and immunotherapy, and results in a poor prognosis. Reflective of the key role of metabolism, the number of preclinical and clinical trials about metabolism-targeted therapies for pancreatic cancer is increasing. The poor prognosis of pancreatic cancer patients might be largely improved after employing therapies that regulate metabolism. Thus, investigations of metabolism not only benefit the understanding of carcinogenesis and cancer progression but also provide new insights for treatments against pancreatic cancer.

158 citations

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