estrogen action, bone cell function, and osteoporosis

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






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






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






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


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


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




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




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



glw227slaP3




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




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


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


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


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


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


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

estrogen action, bone cell function, and osteoporosis

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