Gene Expression Analysis Reveals the Complexity of Pancreatic Disease

Gene Expression Analysis Reveals the Complexity of Pancreatic Disease

Craig D. Logsdon, Ph.D.Professor of Cancer Biology

University of Texas, MD Anderson Cancer Center

Etiological Factors:Etiological Factors:

Acinar EventsAcinar Events

Systemic Inflammatory Response

Systemic Inflammatory Response

Multi-organ Failure- DeathMulti-organ Failure- Death

Local Inflammatory ResponseLocal Inflammatory Response

Temporal and Spatial Complexity of Acute Pancreatitis

Temporal and Spatial Complexity of Acute Pancreatitis

Ethanol abuse Biliary tract stonesTraumaTumorsInfections- mumps, Coxsacie virusDrugs- tetracyclineMiscellaneous (scorpion bites, etc)Idiopathic Hereditary

Multiplicity of Processes Occuring During of Acute Pancreatitis

Multiplicity of Processes Occuring During of Acute Pancreatitis

• Increased Severity– Acinar cell necrosis– Chemoattraction activation of leukocytes– Edema- vasodilation and increased vascular

permeability– Neurogenic inflammation

• Reduced Severity/Recovery– Acinar cell apoptosis– Increased chaperone expression (HSPs) – Regeneration– Fibrosis

• Increased Severity– Acinar cell necrosis– Chemoattraction activation of leukocytes– Edema- vasodilation and increased vascular

permeability– Neurogenic inflammation

• Reduced Severity/Recovery– Acinar cell apoptosis– Increased chaperone expression (HSPs) – Regeneration– Fibrosis

PancreatitisBlind Men and the Elephant

Moral:So oft in theologic wars,

The disputants, I ween,Rail on in utter ignorance

Of what each other mean,And prate about an Elephant-Not one of them has seen!

John Godfrey Saxe (1816-1887)

Specific characteristics of acute pancreatitis are mediated by the expression of critical genes.

Specific characteristics of acute pancreatitis are mediated by the expression of critical genes.

Overarching Hypothesis:

Gene Expression Determines the Outcome of Acute Pancreatitis

Overarching Hypothesis:

Gene Expression Determines the Outcome of Acute Pancreatitis

C vs NC vs NCaerulein PancreatitisCaerulein Pancreatitis

Selection criteria: fold >3 p

C vs NC vs N

C vs JMVC vs JMV

Caerulein PancreatitisCaerulein Pancreatitis

Selection criteria: fold >3 p

C vs NC vs N

C vs JMVC vs JMV

Caerulein PancreatitisCaerulein Pancreatitis

Selection criteria: fold >3 p

in vitro vs Nin vitro vs NC vs NC vs N

127127

C vs JMVC vs JMV

Caerulein PancreatitisCaerulein Pancreatitis

Selection criteria: fold >3 p

Ji, B. X-Q Chen, D.E. Misek, R. Kuick, S. Ernst, R. Najarian, and C. D. Logsdon. Pancreatic Gene Expression during the Initiationof Acute Pancreatitis: Identification of EGR-1 as a Key Regulator.Physiol Genomics 14(1):59-72, 2003

1. Caerulein (20ug/kg ip)2. Taurocholate (50 ul 1%) retrograde3. Controls- JMV180, saline 4. 78 novel genes

Microarray Profiling of Caerulein and Taurocholate

Rat Acute Pancreatitis

Several Functional Categories:Gene RegulatorsSecreted FactorsStructural ProteinsStress Related MoleculesOthers

NFκB Activation Explains Several of the Induced Genes

NFκB Activation Explains Several of the Induced Genes

Many are pro-inflammatory

Multiple Gene Regulators Are Rapidly Activated During Acute PancreatitisMultiple Gene Regulators Are Rapidly Activated During Acute Pancreatitis

1. Many transcription factors and other gene regulators are rapidly induced.

2. Most peak early-unlikely down-stream of trypsin activity

3. Some are pro- others are anti-inflammatory

Acute Pancreatitis Involves The Generation of Opposing MoleculesAcute Pancreatitis Involves The

Generation of Opposing Molecules

ProinflammatoryGro α (KC)

IP-10 Interleukin 6 (IL-6)

Small inducible gene JE (MCP-1)

ProinflammatoryGro α (KC)

IP-10 Interleukin 6 (IL-6)

Small inducible gene JE (MCP-1)

Anti-inflammatoryIκBα (NFκB inhibitor)Tis 11, 11b (cytokine mRNA destabilizers)Lipocortin II (inhibits phospholipase A2)Pancreatitis-associated protein I, III

Anti-inflammatoryIκBα (NFκB inhibitor)Tis 11, 11b (cytokine mRNA destabilizers)Lipocortin II (inhibits phospholipase A2)Pancreatitis-associated protein I, III

Pro-coagulation and FibrinolysisPlasminogen activator, tissue

Tissue Factor

Pro-coagulation and FibrinolysisPlasminogen activator, tissue

Tissue Factor

Anti-coagulation and FibrinolysisPAI-1 (plasminogen activator inhibitor)Anti-coagulation and FibrinolysisPAI-1 (plasminogen activator inhibitor)

Protease inhibitorsLatexin (inhibits metallocarboxypeptidases)Timp-1 (tissue inhibitor of metalloproteinase)

Protease inhibitorsLatexin (inhibits metallocarboxypeptidases)Timp-1 (tissue inhibitor of metalloproteinase)

Proteasespreprotrypsinogen IV

Proteasespreprotrypsinogen IV

Severity of Pancreatitis

Greater Lesser

What About a More Severe Model?

saline ip (20x)

arginine ip, 4hours (20x) arginine ip, 8hours (20x) arginine ip, 24hours (20x)

ER Stress Target Genes are Induced in Acute PancreatitisER Stress Target Genes are Induced in Acute Pancreatitis

Unigene TitleActivating transcription factor 3reticulocalbin 2Myeloid differentiation primary response gene 116UDP glycosyltransferase 1 family, polypeptide A7UDP glycosyltransferase 1 family, polypeptide A6brain glucose-transporter protein metallothionein-2 and metallothionein-1 genes, complete cdsmetallothionein 1Heat shock 27 kDa proteinferredoxin 1GADD45alphaHeat Shock Protein 86

Many other ER stress targets are induced byphysiological concentrations of secretagogues!!

The Pancreatic Acinar Cell is a Professional Secretory Cell

The Pancreatic Acinar Cell is a Professional Secretory Cell

Digestive Enzymes:Synthesize Store Secrete

Secreted molecules are synthesized in the endoplasmic reticulum (ER)

Candidate for ER stress?

Digestive Enzymes:Synthesize Store Secrete

Secreted molecules are synthesized in the endoplasmic reticulum (ER)

Candidate for ER stress?

ER

ZG

ER Stress ResponseER Stress Response

Perk

ER Stress

ATF6 IRE1

XBP1

Apoptosis

Chop Caspase Activity

JNK

Transcription FactorKinase

Protective Molecules

Stress Adaptation

ATF4

ERAD Components

Translationalattenuation

Chaperones

Amino AcidMetabolismAntioxidants

ATF3

Determine the Relationship Between Secretagogue Induced Acute Pancreatitis

and ER Stress Mechanisms

Determine the Relationship Between Secretagogue Induced Acute Pancreatitis

and ER Stress Mechanisms

Compare the effects on ER stress responses of treating pancreatic acinar cells isolated from rats with different concentrations of secretagogues that:

Cause pancreatitis: CCKDo not cause pancreatitis: JMV180, bombesin

Compare the effects on ER stress responses of treating pancreatic acinar cells isolated from rats with different concentrations of secretagogues that:

Cause pancreatitis: CCKDo not cause pancreatitis: JMV180, bombesin

All Secretagogues Induced BiP But Only CCK Induced CHOP in Pancreatic Acini

All Secretagogues Induced BiP But Only CCK Induced CHOP in Pancreatic Acini

BIP

CCK-8 bombesin JMV-180control

control

RT-PCRCHOP*

CCK-8 bombesin JMV-180

*

*

* * * **“ER Stress” is normalFor acinar cells

Severe ER stress is associated with acute pancreatitis

BiPBiP

actinactin

argininesaline

1 4 8 24 72 1 4 8 24 72 h

Arginine Treatment Induces a Rapid Induction of BiP and Chop

Summary:Pancreatitis and ER Stress

Summary:Pancreatitis and ER Stress

ER stress mechanisms are activated by physiologic stimulation of acinar cells

Excessive ER stress is observed only with treatments associated with pancreatitis= caerulein and arginine

ER stress mechanisms are activated by physiologic stimulation of acinar cells

Excessive ER stress is observed only with treatments associated with pancreatitis= caerulein and arginine

ConclusionsConclusions

Multiple mechanisms are involved in the initiation, propagation, and resolution of acute pancreatitis.

NFκB is but one of several transcription factors and gene regulators involved in activating the inflammatory response and in determining mechanisms of cell death.

ER stress mechanisms are important in normal pancreatic physiology and are also involved in acute pancreatitis. Whether they are primarily protective or damaging or both is currently unknown.

Multiple mechanisms are involved in the initiation, propagation, and resolution of acute pancreatitis.

NFκB is but one of several transcription factors and gene regulators involved in activating the inflammatory response and in determining mechanisms of cell death.

ER stress mechanisms are important in normal pancreatic physiology and are also involved in acute pancreatitis. Whether they are primarily protective or damaging or both is currently unknown.

Acute PancreatitisAcute Pancreatitis

InitiatingFactorsInitiatingFactors

Rapid Responses(Acinar Cells)

Rapid Responses(Acinar Cells)

Gallstones

Ethanol abuse

Hyper-stimulation

Hereditary factors

Altered Cell SignalingTrypsin Activation Cytoskeletal Disruption,ER Stress

Activation of Stress associated TFs

Altered Gene Expression:1) Gene Regulators 2) Target Genes

Long TermResponsesLong TermResponsesPancreas

NecrosisApoptosisEdemaInflammation HemorrhageRecovery

Systemic Inflammation

LungRenal

The Logsdon LabLogsdon Lab Baoan JiThiruvengadam ArumugamVijaya Ramachandran Maren FuentesJian SongTobias GroteSebastian GaiserXue PanZobeida Cruz-MonserrateJennifer HsingPavel LevinChantale CharoFadi Braiteh

Logsdon Lab Baoan JiThiruvengadam ArumugamVijaya Ramachandran Maren FuentesJian SongTobias GroteSebastian GaiserXue PanZobeida Cruz-MonserrateJennifer HsingPavel LevinChantale CharoFadi Braiteh

MD Anderson Cancer CenterRosa Hwang, Surgery

Todd MooreArmando Rivera

Hua Wang, PathologyConstanzeKubisch Xue-quingChen