presentation to the Physiological Society Symposium on Epithelial Membrane Transport, Newcastle-upon-Tyne, September 2009

1. Anti-secretory Action of Estrogen in the Intestine
Brian Harvey
Department of Molecular Medicine
Royal College of Surgeons in Ireland
The Physiological Society Membrane and Epithelial Transport Symposium
Newcastle-upon-Tyne 2009

2. RCSI Molecular Medicine
Estrogen rapid responses
Rodrigo Alzamora
Fiona O’Mahony
Warren Thomas

3. Estrogen and Fluid Retention
O’Mahony & Harvey J Physiol 2009

4. Anti-secretory action of Estrogen
X
PKC
Condliffe, Doolan & Harvey J Physiol 2001 O’Mahony & Harvey Mol Endo 2008
O’Mahony & Harvey JBC 2007 O’Mahony et al. Mol Endo 2009

5. Estrogen Inhibits Secretion in Colonic Epithelia
control
E2
1nM E2
Condliffe, Doolan, Harvey J Physiol, 2001.

6. Estrogen Inhibits Secretion via PKC
Rottlerin
E2 -PKC
E2
Condliffe, Doolan, Harvey J Physiol 2001.

7. Estrogen Rapidly Inhibits KCNQ1
Currents in Single Isolated Crypts
1nM
O’Mahony & Harvey J Biol Chem, 2007.

8. Estrogen Phosphorylation of the KCNQ1 Channel
is PKC and PKA dependent
-PKC -PKA
E2
E2 E2
O’Mahony & Harvey J Biol Chem, 2007.

9. Summary
Estrogen causes a rapid and sustained inhibition of
both Ca2+ and cAMP driven chloride ion secretion
via PKC - PKA phosphorylation of KCNQ1 channels
causing closure and retrieval of the channel
Estrogen inhibits basal secretion and secretion activated
by cholera toxin and heat-stable enterotoxin
Questions
 Is the estrogen receptor involved?
 Is the response female-specific?

10. A plasmamembrane-associated 66kDa mER
transduces E2 activation of PKC
O’Mahony et al. Mol Endo 2009.

11. Anti-secretory E2 response is female
sex-specific and estrus cycle dependent
Cl- secretion
rate
% Isc
O’Mahony et al. Mol Endo 2009.

12. Sex differences in ion transporter expression
NKCC1 KCNE3
Male Female Male Female
CFTR Na/K pump
O’Mahony and Harvey. Steroids, 2008.

13. Female sex-specific activation of PKC by E2
Male
Female
O’Mahony et al. Mol Endo 2009.

14. Rapid and Genomic Ion transporter Responses to E2
Apical Basolateral
Short term anti- Na+
secretory effect NKCC1 K+
2Cl-
CREB
MAPK
Transcriptional
PKCAI
mER
priming PKC  PKCAI E2
P
Intracellular
Long term anti- PKC  pool KCNQ1 K+
secretory effect Endocytosis
Cl- CFTR
K+
Exocytosis
Na/K
ATPase
Na+
O’Mahony & Harvey. J Physiol 2009.

15. Summary
 Estrogen regulates Cl- secretion
via PKC inhibition of KCNQ1 channels
 The anti-secretory response is female sex-specific
and estrus cycle dependent
 Estrogen regulates the expression of protein kinases
and ion transporters to effect sustained inhibition of secretion
 A membrane-bound 66KDa mER mediates
the rapid kinase responses to estrogen and genomic priming
of the anti-secretory response

16. mERrole in ion transport and cell proliferation
mER
KCNQ1
Ion transport
K+ response
Cell proliferation
response
mER
O’Mahony & Harvey. Steroids 2008.

17. MAPK Expression is Gender Regulated
Male Female Male Female
O’Mahony et al. Mol Endo 2009.

18. CREB is Phosphorylated in the nucleus in Response to E2
And is MAPK and PKA Dependent
O’Mahony et al. Mol Endo 2009.

19. Rapid mER-estrogen regulated transcriptional responses
O’Mahony et al. Mol Endo 2009.

20. Conclusions
• Estrogen inhibits epithelial ion and fluid secretion in females
only
• The anti-secretory response to estrogen has both beneficial
and pathological effects in intestine and lung, respectively
• The KCNQ1:KCNE3 channel is differentially regulated by
estrogen in females
• The mER receptor transduces rapid anti-secretory
responses to estrogen and primes the genomic phase of
expression of protein kinases, ion channels and receptors,
and cell proliferation

21. The molecular mechanisms for estrogen anti-secretory responses
are similar in colon and bronchus
mER
PKC PK
 A
MAPK
CREB
PKC mRNA
Dehydration of mucus layer
Estrogen in CF, asthma ??

22. Estrogen and KCNQ1 channels
in CF and Asthma?

23. Respiratory function in Asthma and Cystic Fibrosis
is exacerbated in females at puberty - role for E2?

24. A Estrogen reduces ASL in CF bronchus
CuFi_1
55
1H 4H 12H 24H
50
45
40
CuFi control
35
ASL High m m
CuFi_1
30 CuFi + E2
NHBE
25
20
15
10
5
ASL stabilized
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Hours

25. Summary
 The estrous cycle differences in the anti-secretory
response to estrogen may be important for fluid retention
and volume expansion required for implantation of the
embryo
 The anti-secretory effects of estrogen are observed in
other epithelia such as endometrium, sweat gland, renal
CCD and bronchus.
 Estrogen modulation of KCNQ1 channel activity has
important physiological implications for female-specific
responses in secretory diarrhoea, Cystic Fibrosis,
Asthma ….