Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.

1. Paloma Pérez
Laboratorio de Modelos Animales de Patologías Cutáneas
Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas
(IBV-CSIC) Valencia (Spain)
International symposium “Rare skin diseases: from clinic to gene and vice versa”
Madrid 2016
Novel molecular mechanisms of glucocorticoid action:
implications for treating skin diseases

2. Vandevyver
et al., 2014
GLucocorticoid Response Element
GRE
GR and MR share high similarity in structure/function.
Both are ligand-dependent TFs that belong to the NHR family, can
MR/MR) or hetero-dimers (GR/MR), and recognize the same Horm
GR
MR
The glucocorticoid receptor (GR)
HSP90
HSP90
HSP90
HSP90
Glucocorticoid Response
Element (GRE)

3. Keratinocyte-targeted overexpression of the glucocorticoid receptor in mice
(K5-GR mice) induces severe epithelial defects
K5-GR
rGR Poly A
K5-GR
b-globin
Intron
A5.2 kb
Krt5 regulatory sequences
ba c
CO
a
b
c
b´ c´
a´
b´
c´
K5-GR
a´
Donet et al., Mol Endo 2008
CO K5 GR
Pérez et al., FASEB J 2001
CO
K5 GR

4. K5-GR mice: a model for studying Ectodermal Dysplasia
Cascallana et al., ENDO 2005
CO K5-GR
CO K5-GR

5. Impaired NF-kB activity and p63 expression in several K5-GR epithelia
CO K5-GR CO K5-GR
Cascallana et al., ENDO 2005

6. *
% BrdU-positive cells
CO
0
5
10
15
20
25
GREKO
K6LOR
P0 CO GREKO
Sevilla et al., 2013
E17.5
ACTIN
GR
CO GREKO
Newborn Epidermis
GR Epidermal Knock-Out/GREKO mice:
Epidermal inactivation of the GR triggers skin barrier defects
CO GREKO

7. Gene symbol Gene description Fold-change
Keratinocyte proliferation/differentiation
Krt6a keratin 6A 13
Krt6b keratin 6B 8
Krt16 keratin 16 5,9
Krt77 keratin 77 5,2
Sprr2e small proline-rich protein 2E 7,4
Sprr2f small proline-rich protein 2F 6,6
Sprr2h small proline-rich protein 2H 5,0
Sprr2b small proline-rich protein 2B 3,7
Sprr2d small proline-rich protein 2E 3,1
Sprr2i small proline-rich protein 2I 2,6
Sprr1b small proline-rich protein 1B 2,4
Sprr1a small proline-rich protein 1A 2,2
Sprr3 small proline-rich protein 3 2,2
Sprr2k small proline-rich protein 2K 1,6
Lce3a late cornified envelope 3A 8,4
Lce3f late cornified envelope 3F 6,7
Lce3e late cornified envelope 3C 2,6
Rptn repetin 5,7
Pglyrp3 peptidoglycan recognition protein 3 2,6
S100a9 S100 calcium binding protein A9 3,6
S100a8 S100 calcium binding protein A8 2,1
Elf5 E74-like factor 5 3,4
Gene symbol Gene description Fold-change
Peptidase and peptidase inhibitor activity
Slpi secretory leukocyte peptidase inhibitor 3,5
Mmp3 matrix metallopeptidase 3 2,8
Klk6 kallikrein related-peptidase 6 2,9
Klk12 kallikrein related-peptidase 12 2,1
Klk9 kallikrein related-peptidase 9 2,0
Klk10 kallikrein related-peptidase 10 1,9
Adam8 a disintegrin and metallopeptidase domain 8 1,8
Serpina3h serine peptidase inhibitor, clade A, member 3H 0,5
Serpinb3c serine peptidase inhibitor, clade B, member 3C 0,3
Immune response
Tslp thymic stromal lymphopoietin 2,6
Ifi202b interferon activated gene 202A 4,7
Cxcr2 chemokine (C-X-C motif receptor) 3,7
Cxcl16 chemokine (C-X-C motif) ligand 16 1,8
Il33 interleukin 33 2,6
Ereg epiregulin 2,9
Bcl3 B-cell leukemia 3 3,3
Defb1 defensin beta 1 0,6
Tnc tenascin C 2,5
Psors1c2 psoriasis susceptibility 1 candidate 2 3,3
Stat3 signal transducer and activator of transcription 3 1,3
Bcl3
Elf5
Ereg
Fkbp51
krt77
Mmp3
Slpi
Stat3
S100a8
S100a9
Tslp
0,1
1
10
100
RelativemRNAlevels
GREKO/CO
P0epidermis
• High overlapping patterns of gene dysregulation
among GREKO newborn epidermis and the skin of
patients suffering from atopic dermatitis and psoriasis
Gene expression changes indicate
an intrinsic epidermal stress response of GREKO mice to barrier disruption

8. P0
GREKOCO
CO GREKO
p-STAT3
STAT3
p-AKT
AKT
ACTIN
S100A9
0
50
100
150
200
250
300
350
pSTAT3/
STAT3
pAKT/
AKT
S100A9
*
*
*
CO
GREKO
• GREKO skin phenotype resolved
spontaneously around P5
*
P2 P5 COGREKO
K5 FIL LOR
• GREKO adult skin shows mild defects
GREKO skin phenotype resembles AD/psoriasis

9. Ligand-activated GR induces terminal differentiation in primary keratinocytes
Control Dex 24hB
Control Dex 2h
GR
A
C
*
* **
0
2
4
6
8
10
12
24h Dex
24h Ca2+
Sprr2dCdsn

10. • ChIP-seq analysis identified 123 peaks with GR binding upon GC treatment in MPKs (Dex 2h)
Sevilla et al.,MCE 2015
In collaboration with Ian Mills, EMBL Center for Molecular Medicine Norway, Univ. of Oslo
Identification of GR primary transcriptional targets in keratinocytes
http://sartorlab.ccmb.med.umich.edu/chip-enrich
A
B
0
10
20
30
**
**
*
*
*
**
*
C
1
10
100
1000
** **
** **
***
*

11. Validation of GR ChIP-seq data in adult mouse keratinocyte cell lines
GREKOCO
Vehicle
Dex
*
0
1
2
3
4
5
6
7
8
GREKO GREKOCO CO
Tsc22d3 Zfp36
* **
siRNA: CO Klf4
Klf4
GR
TUB
*
** **
**
Klf4
Tsc22d3
Zfp36
0
1
2
3
4
5
6
7
8
CO Klf4 siRNA
Dex - + - +
**
*
0
10
20
30
40
50
60
Tsc22d3
Zfp36
*
**
0
1
2
3
4
5
6
7
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
GR
Tubulin
CO GREKO
• Dex-induced transcription in cultured keratinocytes depends on GR
*
0
1
2
3
4
5
6
7
8
CO
Ts
Klf4
Tsc2
Zfp3
0
1
2
3
4
5
6
7
8
C
Dex -
**
*
0
10
20
30
40
50
60
Tsc22d3
Zfp36
*
**
0
1
2
3
4
5
6
7
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
GR
Tubulin
CO GREKO
GREKO
*
0
1
2
3
4
5
6
7
8
CO
Tsc
Klf4
Tsc22d
Zfp36
0
1
2
3
4
5
6
7
8
CO
Dex -
**
*
0
10
20
30
40
50
60
Tsc22d3
Zfp36
*
**
0
1
2
3
4
5
6
7
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
GR
Tubulin
CO GREKO
• Validation of GR ChIP-seq data in adult mouse keratinocyte cell lines
*
0
1
2
3
4
5
6
7
8
CO
Tsc
Klf4
Tsc22
Zfp36
0
1
2
3
4
5
6
7
8
CO
Dex -
**
*
0
10
20
30
40
50
60
Tsc22d3
Zfp36
*
**
0
1
2
3
4
5
6
7
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
GR
Tubulin
CO GREKO *
0
1
2
3
4
5
6
7
8
CO
Ts
Klf4
Tsc2
Zfp3
0
1
2
3
4
5
6
7
8
C
Dex -
**
*
0
10
20
30
40
50
60
Tsc22d3
Zfp36
*
**
0
1
2
3
4
5
6
7
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
GR
Tubulin
CO GREKO
CO

12. Zfp36 peak, chr7:29,161,349-29,161,679
TCCCGCACATTCCGTCCTCGCCGCCCCGCCCCACCCCGCCCTCCTTCCTTGGCCCTGTGGGGACGGAA
ACATCCCGTTCCTGCCCGAGCTGGGTCAAGAGCCGGAGGGACAGGACCAGAGCACCCCTTAC
GR bound sequences containing KLF sites 40%
GR bound sequences containing AP-1 sites 25%
GRE KLF
KLF
Vehicle
Dex
GRE KLF AP-1
*
Vehicle
Dex
Tsc22d3 peak, chrX:137,063,762-137,064,002
GGGCAAAAAACAGAATGTTCAGCATTCAAGGGAGGGGCAGGAGTTGGGTTCTGCCTGAGTAATTCTTG
GR bound sequences in Dex-treated keratinocytes contained GRE, KLF, and AP-1 sites
GRE
A
*
Vehicle
Dex
Tsc22d3 peak, chrX:137,063,762-137,064,002
GGGCAAAAAACAGAATGTTCAGCATTCAAGGGAGGGGCAGGAGTTGGGTTCTGCCTGAGTAATTCTTG
GRE
B
Vehicle
Dex
Zfp36 peak, chr7:29,161,349-29,161,679
TCCCGCACATTCCGTCCTCGCCGCCCCGCCCCACCCCGCCCTCCTTCCTTGGCCCTGTGGGGACGGAA
ACATCCCGTTCCTGCCCGAGCTGGGTCAAGAGCCGGAGGGACAGGACCAGAGCACCCCTTAC
GRE sites 60%
GC-induced leucine zipper
Gilz/Tsc22d3
Tristetraprolin
Ttp/Zfp36
Software to identify
TF binding motifs MEME
in GR ChIP-seq peaks

13. Antecents of cooperative actions of GR and KLF4
Patel et al., PNAS 2006
However, there was not previous evidence of direct co-regulation of keratinocyte gene expression by GR
and Klf4, in part due to the absence of a suitable model to evaluate GR-mediated gene regulation
• GC treatment of developing K5-Klf4 mice further accelerated formation of the epidermal barrier
• KLF4 plays a key role in skin homeostasis
• Overlapping between gene subsets regulated by GC treatment or Klf4 overexpression

14. GR and KLF4 cooperate to modulate transcription on a subset of
GC-regulated genes in keratinocytes
Vehicle
Dex
*
0
1
2
3
4
5
6
7
8
GREKO GREKOCO CO
Tsc22d3 Zfp36
* **
siRNA: CO Klf4
Klf4
GR
TUB
*
** **
**
Klf4
Tsc22d3
Zfp36
0
1
2
3
4
5
6
7
8
CO Klf4 siRNA
Dex - + - +
**
*
Tsc22d3
Zfp36
*
**
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
CO GREKO A Vehicle
Dex
*
0
1
2
3
4
5
6
7
8
GREKO GREKOCO CO
Tsc22d3 Zfp36
* **
siRNA: CO Klf4
Klf4
GR
TUB
*
** **
**
Klf4
Tsc22d3
Zfp36
0
1
2
3
4
5
6
7
8
CO Klf4 siRNA
Dex - + - +
**
*
Tsc22d3
Zfp36
*
**
Tsc22d3/Gilz
Zfp36/Ttp
CO GREKO
CO GREKO
B
##

15. Dex (min) - 30 60 - - 30 60
Klf4
p-GR
GR
Input RIgG Klf4 IP
GR/KLF4 physical interaction in keratinocytes?
GRE KLF
*
Vehicle
Dex
Tsc22d3 peak, chrX:137,063,762-137,064,002
GGGCAAAAAACAGAATGTTCAGCATTCAAGGGAGGGGCAGGAGTTGGGTTCTGCCTGAGTAATTCTTG
?

16. 0
20
40
60
80
100
120
140
CO
GREKO
*
• Klf4 gene expression is decreased
in GREKO vs control cultured keratinocytes
Does GR induce Klf4 transcription in keratinocytes?
A
• However, no Klf4 mRNA up-regulation nor
GR recruitment to various Klf4 genomic
sequences was detected in Dex-treated
control keratinocytes
0
20
40
60
80
100
120
140
control Dex 30min Dex 1h Dex 3h
Dex - 30 min 1h 3h
B
0
2
4
6
8
10
12
14
16
Klf4 promoter
containing GREs
(macrophages)
Other Klf4
binding sites
Tsc22d3
C

17. B
0.1
1
10
100
Sprr2d Nr3c1
/Gr
Klf4 Tsc22d3 Zfp36
CO
**
**
**
*
CO t0 GREKO t0
CO t72 GREKO t72
A
*
*
*
CO t0
CO t72
GREKO
*
GREKO t0
GREKO t72
DNp63
Tubulin
DNp63
**
*
GREKO t0 GREKO t72CO t0 CO t72
Klf4
GR
C
CO t0
CO t72
GREKO t72
GREKO t0
0
1
2
3
4
5
6
7
GR Klf4
*
**
D
Aberrant regulation of KLF4 and p63 in GR-deficient keratinocytes

18. GR regulates p63 isoform expression
*
**
**
**
DNp63 TAp63
A
0
1
2
3
4
5
6
7
8
9
10
CO t0
CO t72
GREKO t72
GREKO t0
Keratinocytes at low and high calcium
0
0.5
1
1.5
2
2.5
3
DNp63 TAp63
CO V
CO Dex
GREKO Dex
GREKO V
**
*
B
Keratinocytes incubated with vehicle or Dex

19. p63 may be a transcriptional target of GR
*
Vehicle
Dex
A
• GR is recruited to genomic regions nearTrp63
B
FoldrecruitmentGR
**
0
5
10
15
20
25
30
V Dex 2h
Sevilla et al., unpublished

20. Latorre et al., JID 2013
• GREKO keratinocytes exhibited abnormal morphology
resembling partial epithelial-mesenchymal transition (EMT) phenotype
GREKO keratinocytes exhibit features of EMT
0
1
2
3
4
5
Relativeexpression
GR
E-CAD
SMA
K5
TUBULIN
CO GREKO
SNAIL
SLUG

21. Reinsertion of GR in GREKO cells not enough to restore normal keratinocyte morphology
0
1
2
3
4
5
6
7
CO GREKO/EV GREKO/GR
Dex - + - + - +
Nr3c1/Gr
Gilz
V Dex
GREKO/EVGREKO/GR
Dex - + - +
GR
ACTIN
KLF4
p63
GREKO/EV GREKO/GR
Sevilla et al.,
unpublished
GR
ACTIN

22. t0 t72 t72 RU486
**
A
0
40
80
120
160
t0 t72 t72 RU486
*
Sprr2dexpression
(relativetot0)
0
1
2
3
t0 t72 t72 RU486
*
Klf4expression
(relativetot0)
B
Impaired keratinocyte differentiation in the presence of a GR antagonist
CO keratinocytes

23. KLF4 overexpression partially reverses morphological changes (EMT)
of GR-deficient keratinocytes
B GREKO/EV GREKO/Klf4
t0t72
Sevilla et al., unpublished
EV KLF4
E-CAD
p63
ACTIN
KLF4
GREKO
A

24. GR: a master regulator of keratinocyte differentiation?
Impaired Differentiation
Excessive Inflammation
Diseased skin
Differentiation
Inflammation
Healthy skin
GREKOCO
GR GR
p63 p63
Klf4
Klf4
GREnnnnnnKLF Tsc22d3/Gilz
Klf4
GREnnnnnnKLF Tsc22d3/Gilz
GR Klf4

25. Summary
• The generation of mouse models with keratinocyte-specific GR-gain- and loss-of-function
demonstrates that this TF plays a crucial role in epidermal development
• GR regulates the expression of many genes that are key in epidermal differentiation
(including genes of the EDC)
• Transcriptional actions of GCs in keratinocytes involve cooperation between GR and KLF4
• GR modulates the expression and function of KLF4 in keratinocytes, likely due to a direct
transcriptional regulation of the KLF4 repressor, p63
• GR inactivation in keratinocytes correlates with an epithelial-mesenchymal transition (EMT)
phenotype that are partially reversed by modulating the relative levels of GR, p63, and
KLF4 as well as their functional interactions

26. Paloma Pérez
Laboratorio de Modelos Animales de Patologías Cutáneas
Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Científicas
(IBV-CSIC) Valencia (Spain)

28. And Grhl2?
Mehrazarin et al., JBC 2015
Sevilla et al., unpublished
*
Vehicle
Dex
• GR is also recruited to genomic regions near Grhl2
(containing two putative GRE sites)