SIAM Life Science 2012Session MS40Signaling: Vitamin D in Monocytes andReceptor Clustering in Mast Cells

Hewison and Adams laboratoryUCLA-Orthopaedic Hospital

Research CenterRene Chun

August 9, 2012

Monocyte Innate Immune Response toVitamin D

Objectives

• Background on vitamin D and humanhealth

• Overview of vitamin D metabolism andaction, in particular, the role of serumvitamin D binding protein (DBP)

• Some findings on vitamin D and immunefunction and DBP

• Development of mathematical models• Implications from mathematically analysis

Vitamin D Supplements

Historical: Childhood Rickets Due toLow Vitamin D

Hewison and Adams labs at UCLA

Vitamin D

Public health significance:Some (many?) may have vitamin D levels too low to actualizeits full benefits

Current: Areas of Interest

bonecomplications

duringpregnancy

inflammatory bowel

disease

kidney disease

immune responseheart disease

Forms of vitamin D

OH

OH

1,25(OH)2D3

1α-hydroxylase

OH

OH

OH

1,24,25(OH)3D3

24-hydroxylase

OHOH

24,25(OH)2D3

1α-hy

droxyl

ase

24-hydroxylase

OH

25(OH)D3

25-hydroxylase

vitamin D3

7-dehydrocholesterolU.V.B

photolysis

(CYP2R1)

(CYP27B1) (CYP24A1)

Bone healthImmune regulationAnti-proliferation

Vitamin D and Bacterial Killing

DBP polymorphisms

GC-‐1S

GC-‐1F

GC-‐2

glu-‐ala-‐thr-‐pro-‐thr

asp-‐ala-‐thr-‐pro-‐thr

asp-‐ala-‐thr-‐pro-‐lys

1F > 1S > 2 affinity for 25D

Arnaud J, et al., Hum Genet. 1993 Sep;92(2):183-8

White GC-1S > GC-1FBlack GC-1F > GC-1SWhite GC-2 > Black GC-2USA Blacks

GC-1F 70%GC-1S16%GC-2 11%

USA WhitesGC-1F 17%GC-1S55%GC-2 26%

Since affinity for 25D: GC-1F > GC-1S > GC-2 Perhaps a basis for some differences between whites and blacks in vitamin D influenced health outcomes?

DBP and Ethnic VariationsKamboh, Human Genetics (1986) 72:281-293

JCEM 95:3368 (2010)

Questions

Did bound or free-ligand entry drive response?

How did DBP polymorphisms impact response?

JCEM 95:3368 (2010)

Findings

Did bound or free-ligand entry drive response?Free-ligand entry

How did DBP polymorphisms impact response?Lower affinity DBP yielded higher response

How much free ligand?

25D  +  DBP

25D  +  ALB

1,25D  +  DBP

1,25D  +  ALB

25D/DBP

25D/ALB

1,25D/DBP

1,25D/ALB

Ka  (M-‐1)

7x108

4x107

6x105

5.4x104

How much free ligand?

Mathematical Analysis

Collaborators:• Brad Peercy, Dept of Mathematics & Statistics,

Univ of Maryland, Baltimore County• Arthur Sherman, Lab of Biological Modeling,

NIDDK, NIH

Mathematical Analysis

Expand existing mathematical model by adding ….• Multiple genotypes for DBP (model #1)• In vitro experimental data (model #2)

To ask ….Using model #1• How DBP genotype effects free levels of vitamin D ligands?

Using model #2• Does the intracrine or endocrine mechanism account for vitamin D

action in adherent monocytes?

Affinity differences(published):GC1F/GC1FGC1F/GC1SGC1F/GC2GC1S/GC1SGC1S/2GC2/2

Affinity(published):DBP for 25DDBP for 1,25DALB for 25DALB for 1,25D

Concentrations(user input):25D1,25DDBPALB

Variables and parameters

Model #1

Feldman,  Rodbard  &  Levine,  AnalyHcal  Biochemistry,  1972Dunn,  Annals  of  NY  Academy  of  Sciences,  1988

Free Vitamin Levels Based on GenotypicVariation in DBP

PLoS One (2012) 7:e30773

In vitro (5% serum) In vivo

Free Vitamin Levels Based on GenotypicVariation in DBP

PLoS One (2012) 7:e30773

“Sufficiency” as described in Nov 2010report by Institute of Medicine (IOM):20 nanograms per milliliter (50 nM) serum25D is needed for good bone health forpractically all individuals

Model #2

v1  =  25Dv2  =  1,25D

v1

v2

CYP27B1:v1

Albumin:v1

Albumin:v2

DBP:v2

DBP:v1

CYP27B1

VDR:v1

VDR:v2

CAMPDNACAMP

Ka=700µM-‐1

Ka=0.6µM-‐1

Ka=40µM-‐1

Ka=0.054µM-‐1

Ka=5µM-‐1

Km=1µM

Ka=104µM-‐1

intracellular

extracellular

Some knowledge exists …From model #1•Affinity of DBP for 25D & 1,25D•Affinity differences in DBP genotypes•Amounts of DBP in differing genotypes•Affinity of ALB for 25D & 1,25D

Needed for model #2•Affinity of VDR for 1,25D (much less known for 25D)•Amount of VDR in cells (has been estimated in some cell types)•Permeability of cells for 1,25D (none for 25D)•Affinity of CYP27B1 for 25D (reconstitution studies)•Rate of conversion to 1,25D by CYP27B1 (reconstitution studies)

Guesstimated …•Amount of CYP27B1•Amount of CYP27B1 and VDR in activated immune cell•Affinity of 25D/VDR binding to VDRE•Affinity of 1,25D/VDR binding to VDRE (EMSA data exists)•Effectiveness of 25D/VDR/VDRE complex on transcription of CAMP•Effectiveness of 1,25D/VDR/VDRE complex on transcription CAMP•Extracellular volume >> intracellular volume

Parameters and variables for model #2

VDR/Ligand Interactions

25D VDR/25D 1,25D VDR/1,25D

r1 r2

r2 > r1

!

r1 =Kr 2

ppv1cmmRT

Kr 2ppv1

cmm + Kr1mmv2

cpp + Kr1mmKr 2

pp, r2 =

K1mmv2

cppRTKr 2

ppv1cmm + Kr1

mmv2cpp + Kr1

mmKr 2pp,

Active Transcription Complexes

o1 = active complex containing 25D

o2 = active complex containing 1,25D

o2 > o1

!

o1 =Kcc2

p r1m

Kcc1m r2

p + Kcc2p r1

m + Kcc1m Kcc2

p , o2 =Kcc1

m r2p

Kcc1m r2

p + Kcc2p r1

m + Kcc1m Kcc2

p

Kidney International 56:S46-S51 (1999)

ΔΔCT=log2(CAMP/CAMP0)

CAMP production

…. solve computationallyand then express graphically ….

!

CAMP = " o1 + o2( ) + CAMP0

!

r1 =Kr 2

ppv1cmmRT

Kr 2ppv1

cmm + Kr1mmv2

cpp + Kr1mmKr 2

pp, r2 =

K1mmv2

cppRTKr 2

ppv1cmm + Kr1

mmv2cpp + Kr1

mmKr 2pp,

!

o1 =Kcc2

p r1m

Kcc1m r2

p + Kcc2p r1

m + Kcc1m Kcc2

p , o2 =Kcc1

m r2p

Kcc1m r2

p + Kcc2p r1

m + Kcc1m Kcc2

p

In vitro (5% serum) data and model fit

Then adjust parameters to project in vivo responses …

Intracrine vs. Endocrine?

Basal = 1x VDR, 1x CYP27B1Activated = 5x VDR, 10x CYP27B1

Summary

Local production of 1,25D from 25D is aplausible mechanism to account for vitamin Daction in immune cells.

DBP genotype, DBP amount and total 25Dinteract to determine free 25D levels which impactsvitamin D action.

Thus, one fixed amount of total 25D to definesufficiency may be an inadequate clinicalguideline.