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ESTROGEN ACTION, BONE CELL FUNCTION, AND OSTEOPOROSIS. ESTROGEN ACTION, BONE AND OSTEOPOROSIS New and Emerging Paradigms. As of 1995, there were five pillars of belief about E-action that were widely accepted by almost everyone - PowerPoint PPT Presentation
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2342
ESTROGEN ACTION,BONE CELL FUNCTION,AND OSTEOPOROSIS
2227
ESTROGEN ACTION, BONE AND OSTEOPOROSISNew and Emerging Paradigms
• As of 1995, there were five pillars of belief about E-action that were widely accepted by almost everyone
• Since then, these have been swept away by a sea change in conventional wisdom
• New paradigms are emerging to replace these traditional concepts
2341
• Molecular bases of E-action
• E-effects on skeletal growth and maturation
• E-deficiency and bone loss in elderly women
• E-interaction with biomechanical strain
• E-deficiency and bone loss in elderly men
ESTROGEN ACTION, BONE AND OSTEOPOROSISAreas of Paradigm Shifts
2105.1aP1
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
1. E-action on the skeleton
a) is transduced through a single ER in bone cells
b) is gender-specific for its receptor
c) is mediated by a single, or only a few, paracrine cytokines
2105.1aP2
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
1. E-action on the skeleton
a) is transduced through a single ER in bone cells
b) is gender-specific for its receptor
c) is mediated by a single, or only a few, paracrine cytokines
2114c
ESTROGEN RECEPTORS (ERs) IN BONE CELLSRecent Advances
• Discovery and cloning of 2nd ER (PNAS 93:5925, 1996)
• ER is always a transcriptional activator whereas ER is sometimes inhibitory (Endocrinology 140:5566, 1999)
• ER predominates in cancellous bone whereas ER predominates in cortical bone (JCEM 86:2309, 2001)
• ER KO mice are protected against age-related loss of cancellous bone (JBMR 165:160, 2001)
• ER/ER ratio may be a key determinate of E-action on bone
2105.1aP3
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
1. E-action on the skeleton
a) is transduced through a single ER in bone cells
b) is gender-specific for its receptor
c) is mediated by a single, or only a few, paracrine cytokines
2340
i. Sex steroids have non-genomic, gender non-specific effects on decr. apoptosis in OB cells and incr. apoptosis in OC cells
• These effects occur via either E or DHT, act througheither ER or AR and can be blocked with either ICI orflutamide (Kousteni et al. Cell 104:719, 2001)
• A synthetic ligand (4-estren-3,7-diol) reproduces thenon-genotrophic effects of sex steroids without affectingclassical transcription and was as effective as either E2 orDHT in increasing bone mass in gonadectomized mice(Kousteni et al. Science, in press)
GENDER NON-SPECIFICITY IN SEX STEROID SIGNALINGRecent Advances
2340.1
ii. At least in the prostate and aorta (and possibly in bone and other target tissues), the androgen metabolite, 5-androstane-3,17-diol (3Adiol) activates signaling via ER (Weihua et al. PNAS 98:6330, 2001)
GENDER NON-SPECIFICITY IN SEX STEROID SIGNALINGRecent Advances (cont’d)
2105.1aP4
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
1. E-action on the skeleton
a) is transduced through a single ER in bone cells
b) is gender-specific for its receptor
c) is mediated by a single, or only a few, paracrine cytokines
2288P1
REGULATION OF OC FORMATION AND FUNCTIONModel
REGULATION OF BONE RESORPTIONRole of Paracrine Cytokines
T CELLSMONOCYTES
ACTIVE OCOC
APOPTOSIS
STROMAL CELLS/OSTEOBLASTS
TGF
TNF
IL-6PGE2
GM-CSF
M-CSF
OC PRECURSORS
TNF
TGF
Differentiationand activation
Stimulatory Factors
Inhibitory Factors
RANKLRANKL
OPG
IL-1TNF
2225b
MEDIATORS OF ESTROGEN ACTIONCandidates
“Upstream” regulatorsIL-1 (Pacifici 1993), TNF (Kimble 1997), IL-6 (Jilka 1992), PGE2 (Kitazawa 1994), IL1ra (Pacifici 1993)
“Downstream” regulators M-CSF (Kimble 1996), OPG (Hofbauer 1999, Saika 2001), TGF (Oursler 1991)
Responsiveness of osteoclasts to RANKLcJUN, NF-B, JNK1 (Shevde 2000, Srivastava 2000)
2293.2
RANKL EXPRESSION IN VARIOUS CELL TYPESEffect of Estrogen Deficiency
60
Marrow stromal cells
90 P<0.001
No
rmal
ized
Flu
ore
scen
ce
Inte
nsi
ty f
or
OP
G-F
c-F
ITC
0
30
B - Cells
P<0.001
T - Cells
P=0.003
Total
P<0.001
PremenopausalN = 12
Untreated PostmenopausalN = 12
Postmenopausal + ERTN = 12
***
** *
** *
***
* Vs PostM E(+) ** Vs PreM
2364
RANKL EXPRESSION PER CELLCORRELATES WITH BONE RESORPTION IN WOMEN
MSC
T-Cells
B-cells
Total
0.48, P=0.002
0.36, P=0.02
0.48, P=0.002
0.36, P=0.02
0.42, P=0.009
0.34, P=0.03
0.48, P=0.002
0.43, P=0.007
Serum CTx Urine NTx
2288P2
REGULATION OF BONE RESORPTIONSites of Estrogen Regulation
T CELLSMONOCYTES
ACTIVE OCOC
APOPTOSIS
STROMAL CELLS/OSTEOBLASTS
TGF
TNF
IL-6PGE2
GM-CSF
M-CSF
OC PRECURSORS
TNF
TGF
Differentiationand activation
Stimulatory Factors
Inhibitory Factors
RANKL
E(-)E(-)
E(-)
E(+)
E(-)E(-)
E(+)
OPG
IL-1TNF
E(+) RANKL
E(-) E(-)
2105.2a
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
1. E-action on the skeleton a) is transduced through a single ER in bone cells; b) is gender-specific for its receptor; and c) is mediated by a single, or only a few, paracrine cytokines
2. Pubertal skeletal growth and maturation is regulated by E in females and by T in males
1170c
• Young adult male with ER gene mutation (Smith 1994) and two young adult males with aromatase gene mutations had low BMD, despite T-sufficiency (Carani 1997, Morishima 1997)
• All three mutants had open epiphyseal growth plates and failed to undergo pubertal growth spurt (Grumbach 1999)
E IS REQUIRED FOR MALES TO ACHIEVENORMAL BMD AND TO CLOSE EPIPHYSES
Results of Human Experiments of Nature
2294
EFFECT OF E TREATMENT ON LINEAR GROWTH AND BMD IN ER AND AROMATASE MUTANT MALES
Aromatase mutant
160
180
Age, yrs
200
Sta
ture
, cm
120
140
Duration of ERT(mo)
ER mutant
8 10 12 14 16 18 20 22 24 26 28
Estrogen
Aro
mat
ase
mu
tan
t
fro
m b
asel
ine,
%
0 12 24 36
0
10
20
0
10
20
0
10
20
LumbarSpine
FemoralNeck
Radius
Smith et al. NEJM 331: 1056, 1994Bilezikian et al. NEJM 339: 599, 1998
220
Normal males (adjusted to pubarche)
2290P1
FEMALEFEMALE
PREPUBERTAL CHILD (8 yr)PREPUBERTAL CHILD (8 yr) YOUNG ADULT (18 yr)YOUNG ADULT (18 yr)
MALEMALE
SEXUAL DIMORPHISM IS ACHIEVED DURING PUBERTY BY DIFFERENTIAL CHANGES ON BONE SURFACES
C.A. = 43 mm2
C.A. = 58 mm2
C.A. = 24 mm2
C.A. = 26 mm2
Garn 1970, Seeman 1999
Gained
Lost
2286
E AND T HAVE BOTH DIVERGENT AND COMPLIMENTARY EFFECTS ON BONE
PERIOSTEAL FORMATION
Turner et al. Endocrinology 122:1146, 1988Turner et al. J. Orthop. Res. 8:612, 1990
60
80
100
120
140 OVX FEMALES ORX MALES
, %
fro
m p
lace
bo
ESTROGEN TREATMENT
TESTOSTERONE TREATMENT
Histomorphometry in 3 mo. old rats 21 days of treatment
BONE RESORPTION
Riggs et al. JCI 51:1659, 1972
Iliac biopsy of PMO women 3 months of treatment
-60
-40
-20
0
, %
res
orp
tio
n s
urf
aces
ANDROGENTREATMENT
P<0.001
P<0.05
ESTROGENTREATMENT
2105.3a
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
1. E-action on the skeleton a) is transduced through a single ER in bone cells; b) is gender-specific for its receptor; and c) is mediated by a single, or only a few, paracrine cytokines
2. Pubertal skeletal growth and maturation is regulated by E in females and by T in males
3. In postmenopausal women, E deficiency causes the early, rapid phase of bone loss, whereas age-related factors cause the late slow phase
glw227sla
PATTERN OF INVOLUTIONAL BONE LOSS
Cortical Bone Cancellous Bone
Menopause Women Men100
90
80
70
60
50
0
100
90
80
70
60
50
050 90807060 50 90807060
Age, yrs Age, yrs
% o
f in
itia
l B
MD
% o
f in
itia
l B
MD
glw227slaP1
PATTERN OF INVOLUTIONAL BONE LOSS
Cortical Bone Cancellous Bone
Menopause Women Men100
90
80
70
60
50
0
100
90
80
70
60
50
050 90807060 50 90807060
Age, yrs Age, yrs
% o
f in
itia
l B
MD
% o
f in
itia
l B
MD
Cortical Loss - 6%Cancellous Loss - 25%
glw227slaP2
PATTERN OF INVOLUTIONAL BONE LOSS
Cortical Bone Cancellous Bone
Menopause Women Men100
90
80
70
60
50
0
100
90
80
70
60
50
050 90807060 50 90807060
Age, yrs Age, yrs
% o
f in
itia
l B
MD
% o
f in
itia
l B
MD
Cortical Loss - 6%Cancellous Loss - 25%
Cortical Loss - 25%Cancellous Loss - 25%
glw227slaP3
PATTERN OF INVOLUTIONAL BONE LOSS
Cortical Bone Cancellous Bone
Menopause Women Men100
90
80
70
60
50
0
100
90
80
70
60
50
050 90807060 50 90807060
Age, yrs Age, yrs
% o
f in
itia
l B
MD
% o
f in
itia
l B
MD
Cortical Loss - 6%Cancellous Loss - 25%
Cortical Loss - 25%Cancellous Loss - 25%
Cortical Loss - 25%Cancellous Loss - 25%
2279a
RAPID TRANSIENT PHASE OF BONE LOSSIS INITIATED BY MENOPAUSE
• Characterized by high bone turnover (BR > BF), predominantly cancellous bone loss and duration of 4 - 8 yr
• Outpouring of Ca from bone partially suppresses PTH secretion
• Caused by loss of restraining effects of E on bone turnover acting directly through ER in OBs and OCs
1220.1
PERFORATIVE RESORPTION OF TRABECULAE IN RAPID PHASE DISRUPTS BONE MICROARCHITECTURE
Micro CT images of vertebral biopsy samplesare courtesy of Ralph Mueller, Ph.D.
Young Normal PostmenopausalOsteoporosis
2280a
THE SUBSEQUENT SLOW PHASE OF BONE LOSS CONTINUES INDEFINITELY
• Pattern of bone loss differs from that of early postmenopausal bone loss
• Similar pattern occurs in aging men
• Driven mainly by 2O hyperparathyroidism associated with age-related impairment of external Ca homeostasis
Thought to be mainly due to age-related abnormalities rather than E-deficiency because
2295
AGING IS ASSOCIATED WITH 2O HPT AND HIGH BONE TURNOVER IN BOTH SEXES
Men
Women
Men Women
64%r=0.30, P<0.001
84%r=0.30, P<0.001
30%r=0.30, P<0.001
62%r=0.25, P<0.001
77%r=0.37, P<0.001
93%r=0.25, P<0.001
Khosla et al JCEM 83: 2266, 1998
2287
EFFECT OF Ca SUPPLEMENTATION OR ERT IN ELDERLY POSTMENOPAUSAL WOMEN
McKane et al JCEM 81: 169, 1996McKane et al Proc Assoc Am Phys 109: 174, 1997
-20
0
20
40
60
%,
you
ng
ad
ult
val
ues Serum PTH Urine free DPD
N=88 (ages 68-78 yrs)
Untreated 1.5 g Ca ERT
-20
0
20
40
60
%,
you
ng
ad
ult
val
ues
P<0.005
P<0.005
P<0.005P<0.005
2226
POSTMENOPAUSAL BONE LOSSQuestions
• How can E-deficiency both suppress PTH secretion (during the early, rapid phase) and increase it (during the slow, late phase)?
• How can both estrogen (a hormone) and calcium (a nutrient) both normalize the increased bone resorption and 2º hyperparathyroidism in elderly women?
The answer lies in the emerging concept of extraskeletal actions of E on peripheral calcium metabolism
NGPP070slB.1
ESTROGEN ENHANCES CALCITRIOL-STIMULATED INTESTINAL Ca ABSORPTIONPerimenopausal women undergoing OVX weretreated with HRT (N=7) or PL (N=7) for 6 mo.
6 months after OVXBSLGennari et al. JCEM 71:1288, 1990
PlaceboHRT
ANOVA P<0.001
1 g/d x7 d Calcitriol
P=0.004
FractionalCa Abs
0
0.3
0.2
0.1
1261A.1
ESTROGEN ENHANCES RENAL Ca CONSERVATION INDEPENDENTLY OF PTH
98
99
100
TRCa, %
97
0
2
3Urine Ca
excretion,µmol/dL GF
1
18 early postmenopausal women before and after 6 mo. of ERT
ERTPTH
**
*#*
*
- -+ +- +- +
* p < 0.001 vs bsl# p < 0.001 forERT vs PTH
*#
McKane et al JCEM 80: 3458, 1995
1280e
ESTROGEN AND BONE FORMATIONBackground
• E increases IGF-I and procollagen production by osteoblasts in vitro (Ernst 1989, Kassem 1997)
• E increases OB differentiation in vitro but effects on proliferation are less established (Harris 1997, Okazaki 2002)
• E increases osteoblast life-span by opposing apoptosis (Manolagas 2000)
• In elderly women, long-term (6-14 yr), high dose estradiol implants increased TBV and WT of trabecular packets (P < 0.001) (Vedi 1994, Khastigir 2001)
CA732660-01C
Indirect effects
Direct effects
Remodelingimbalance Increased
bone resorption
BONE LOSS
Estrogendeficiency(rapid onset)Decreased bone
formationSecondary
hyperparathyroidism
Dietarycalcium
?
INVOLUTIONAL BONE LOSS IN WOMENModel
2106.1
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
4. The two major regulators of bone mass -- sex steroids and biomechanical strain -- have independent actions
2282a
ACTIONS OF E AND MECHANICAL STRAINON BONE CELL FUNCTION ARE RELATED
• Frost (1992) hypothesized that bone contains a cybernetic system (“the mechanostat”) that adjusts bone mass commensurate with mechanical strain
• Because histological changes following immobilization and E-deficiency are similar, Frost (1999) suggested that E deficiency impairs mechanostat sensing
• Osteocytes can sense mechanical strain (Lanyon 1993) and contain ER (Tompkinson 1998)
2283a
ACTION OF E AND MECHANICAL STRAIN ON BONE CELL FUNCTION ARE RELATED (Cont’d)
• Loading studies in E-deficient or E-replete rats before and during orbital space flight (Westerlind 1997) suggest that E and mechanical strain share a common signal transduction pathway(s)
• Effects of mechanical strain on OB proliferation in vitro are blocked by ER antagonists and both E and strain utilize MAP kinase and ERK-1 signaling pathways (The Lanyon Group 1996-2001)
2289
MODEL FOR E AND STRAIN INTERACTIONS
Bone Mass
-
+
Strain signalE(+)
PREMENOPAUSE (Steady State)
Strain generator Mechanostat
E(-)POSTMENOPAUSE(Non-Steady State)
Strain generator Mechanostat
E(-)POSTMENOPAUSE
(Steady State)Strain
generator Mechanostat
0
-
+
0
-
+
0
Bone loss
Bone gain
Strain signalBone loss
Bone gain
Strain signalBone loss
Bone gain
Bone Mass
Bone Mass
2106.2
ESTROGEN ACTION, BONE AND OSTEOPOROSISPrevailing Paradigms (~1995)
4. The two major regulators of bone mass -- sex steroids and biomechanical strain -- have independent actions
5. Sex steroid deficiency plays only a minor role in the etiology of osteoporosis in men
2319
AGE-RELATED BONE LOSS IN MENBackground
• Men lose 1/2 as much bone with aging as women and have 1/3 the number of fractures
• Both sexes have similar patterns of late bone loss and 2º hyperparathyroidism
• Serum total sex steroids decrease only slightly with aging
1172
CHANGES IN SEX STEROIDS OVER LIFE Gender Differences
Lateral spine BMDLateral spine BMD
Serum:Serum:Bio EBio TSHBGLHFSH
*P<0.05, **P<0.005
Men (N=350)Men (N=350)% change% change
-27**
-47**-64**
+124**
+285**
+505**
Women (N=350)Women (N=350)% change% change
-45**
-83**
-28*-1
+731**
+1805**
Khosla, et al. JCEM 83:2266-2274, 1998
1199b
MECHANISMS OF SEX STEROID DEFICIENCYDifferences Between Genders
Onset
E-deficiency
T-deficiency
, SHBG
Mechanism
Females
Begins acutely atmenopause
++++
++
0
Ovarian failure
Males
Gradual and progressive
++
++++
+++
a) Increased binding by SHBGb) Reduced secretory capacity for
GH and T
1518d
RELATIONSHIP OF SERUM E TO BMDResults of Observational Studies
Ten population-based observational studies have now shown that in aging men serum E is more closely related to BMD than is serum T
Slemenda et al. JCI 100:1755, 1997 Greendale et al. JBMR 12:1833, 1997 Khosla et al. JCEM 83:2266, 1998 Center et al. JBMR 15:1405, 2000 Annewieke et al. JCEM 85:3276, 2000 Amin et al. Ann Intern Med 133:951, 2000 Szulc et al. JCEM 86:192, 2001 Goemaere et al. IOF, 2002 Gennari et al. IOF, 2002 Kaufman IMS, 2002
1409.2
EVIDENCE FOR A THRESHOLD FOR BONE LOSS DUE TO E-DEFICIENCY IN ELDERLY MEN
-6
-4
-2
0
2
0 10 20 30Bioavailable E2, pg/mL
Mid
-Rad
ius,
%/y
r
r = 0.05P = 0.702
r = 0.36P = 0.003
0
2
4
6
8
0 10 20 30
nm
ol/
LG
F
r = -0.43P = 0.0004
r = -0.12P = 0.347
Bioavailable E2, pg/mL
Rate of change in BMD Urine NTx
Khosla et al. JCEM 86: 3555, 2001
2026A.2
E RATHER THAN T IS THE MAJOR REGULATOR OF BONE RESORPTION IN ELDERLY MEN
0
10
20
30 ***
***
**
*
40
% change
Urinary DpdUrinary NTx
(-T, -E) (-T, +E) (+T, -E) (+T, +E)
ANOVA E effect P=0.005; T effect P=0.232ANOVA E effect P=0.0002; T effect P=0.085
For changefrom baseline*P<0.05**P<0.01***P<0.001
Falahati-Nini et al. JCI 106: 1553, 2000
CA732660-02F
Indirect effects
Direct effects
Remodelingimbalance Increased
bone resorption
BONE LOSS
Decreased bone formation
Secondaryhyperparathyroidism
Dietarycalcium?
INVOLUTIONAL BONE LOSS IN MENModel
Testosteronedeficiency
Estrogendeficiency(slow onset)
2107c
ESTROGEN ACTION, BONE AND OSTEOPOROSISNew and Emerging Paradigms (2002)
1. E action on the skeleton
a. Is determined in part by ER/ER ratio
b. Includes cross-talk between E and T signaltransduction pathways
c. Involves multiple paracrine mediators but, inhumans, the RANKL/OPG ratio (and possibly
theTNFlevel) in the bone marrow
microenvironmentare particularly important
2108a
ESTROGEN ACTION, BONE AND OSTEOPOROSISNew and Emerging Paradigms (2002)
2. E is the major sex steroid regulating skeletal growth and maturation in both sexes
a. E is an absolute requirement for the pubertalgrowth spurt and for epiphyseal plate closure
b. E contributes substantially to attainment of peakBMD in both sexes
c. Males have larger bones mainly because of divergent actions of T and E on periosteal bone
apposition
2109a
ESTROGEN ACTION, BONE AND OSTEOPOROSISNew and Emerging Paradigms (2002)
3. E deficiency is the major cause of both phases of postmenopausal bone loss
a. Early, rapid phase is caused by loss of direct effects of E on bone cells
b. Late, slow phase is mainly caused by secondaryhyperparathyroidism induced by loss of E
effecton peripheral calcium metabolism
2110b
ESTROGEN ACTION, BONE AND OSTEOPOROSISNew and Emerging Paradigms (2002)
4. The direct effects of E and of biomechanical strain on bone are transduced, at least in part, through a common signaling pathway
5. Bone loss in aging men
a. Caused mainly by combined E and T deficiencydue to both increased SHBG binding andimpaired gonadal secretion
b. BioE is a major predictor but operates only belowa critical threshold level
2320a
• ER was first steroid nuclear receptor to evolve in vertebrates (PNAS 98:567, 2001)
• Birds lose ~30-40% of their bone during egg-laying which is then rapidly restored
• Mammal-like reptiles of Triassic era were oviparous
• In viviparous mammals, this process was co-opted for mineralization of the fetal skeleton and for lactation
• Once in place in females, it could be used to regulate bone mass in males
WHY ARE E EFFECTS ON BONE AND CALCIUM METABOLISM SO COMPLEX?
2119a
OVERALL CONCLUSIONS
• E is the most important sex steroid for the formation and maintenance of the adult skeleton
• Effects of T on bone cells are both complimentary and antagonistic to those of E
• Actions of E are pervasive and involve multiple cytokines and signaling pathways
• Sex steroid deficiency is the most important cause of osteoporosis in both sexes
1270b
ACKNOWLEDGMENTS
StaffStaff
S. Khosla, M.D.
L.J. Melton, M.D.
T.C. Spelsberg, Ph.D.
Postdoctoral fellowsPostdoctoral fellows
G.A. Ledger, M.D.
W.R. McKane, M.D.
A. Falahati-Nini, M.D.
G. Eghbali-Fatourechi, M.D.
Staff of Mayo Osteoporosis Research Group and Mayo GCRCN.I.H. Grants PO1 AG04875 and RO1 AR27065