Effect of an NF-κB Inhibitor on the Cell Population in the

Fibroblast-Populated Collagen Matrix

   Doyle DA, Chao J, Heimann D, Peña T, Hansen C, Carlson MA

University of Nebraska Medical Center

VA Nebraska- Western Iowa Health Care System  

Fibroblast Populated Collagen Matrix Model (FPCM)

attached = mechanically stressed (survival and cell cycle both upregulated è matrix cell population increases) detached = stress-released (survival and cell cycle both downregulated è matrix cell population decreases)

Network  analysis  of  microarray  data  in  a3ached  FPCM    vs.  released  FPCM  suggested  that  NF-­‐κB  

signaling  was  ac?vated  by  matrix  release        

Released  matrix                          NF-­‐κB                

             

                                         

       

Matrix  cell  number    

Hypothesis     NF-­‐κB     Matrix  cell  number    

phospho-p65 (nuclear fraction)

Histone

phospho-IKKα/β (cytosolic fraction)

β-actin

Att 5 10 20 30 60 180 360

Time after matrix release (min)

NF-κB activation occurs after release of the attached 3D FPCM

Methods For Inhibiting NF-κB In FPCM

NF-κB Inhibitor Gel Polymerization Matrix Incubation Detachment Cell Harvest and Count

Gel Polymerization Matrix Incubation Detachment NF-κB Inhibitor Cell Harvest and Count  

Method 1 Method 2

TPCK  and  TPCA-­‐1  inhibit  IKK,  preven?ng  the  ac?va?on  of  NF-­‐κB    

Rel  A,  P50  

IκBα   IKK  

Extracellular  signals    (matrix  release)  

IκBα  

Rel  A,  P50  

P  P  IκBα  

Nuclear  Pore  

P  P  

Proteasome  degrada?on  

Nuclear  DNA    TPCK    TPCA-­‐1  

Inhibi?ng  NF-­‐κB  increases  matrix  cell  number  

TPCK=  N-­‐Tosyl-­‐L-­‐Phenylalanine  Chloromethyl  Ketone    

mean ± sd of 5 experiments; *p < 0.05 compared to {att/72 hr/0 µM}, Kruskal-Wallis

Inhibition of NF-κB activation with TPCA-1 increases matrix cell number in the released matrix at 72 hr (“disinhibition”)

‡2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide, an IkB kinase-2 (IKK-2) inhibitor

TPCA-1‡

* *

Conclusions    

1.  NF-κB is activated after release of the FPCM.

2.  Treatment of the FPCM with small molecule inhibitors of NF-κB activation partially blocked the decrease in cell number which occurs after matrix release.

3.  NF-κB activation likely contributes to the downregulation of matrix cell population after matrix release.