DISCLOSURES PATHOPHYSIOLOGY of OSTEOPOROSIS: Pathways That - - PowerPoint PPT Presentation

PATHOPHYSIOLOGY of OSTEOPOROSIS: Pathways That Control Bone Homeostasis

Dolores Shoback, MD Professor of Medicine, UCSF OSTEOPOROSIS: New Insights in Research, Diagnosis and Clinical Care July 12, 2018

DISCLOSURES

• Nothing to disclose
• No conflicts of interest

TOPICS

• Bone remodeling and modeling

– Imbalances underlie bone loss – Pathways critical in fracture repair and treatments

• Resorption – RANK-L/RANK/OPG
• Formation - Wnt/LRP5/Beta catenin

– Osteoblast, osteoclast, osteocyte

• Role of bone-fat interactions in homeostasis
• Pathogenesis of bone loss
• Menopause
• Aging

BONE REMODELING – coupled resorption and formation that maintains bone mass in adult life

Baron and Hesse, JCEM, 2012

BONE REMODELING – coupled resorption and formation that maintains bone mass in adult life BONE MODELING – process that shapes bones as we grow & develop (childhood, adolescence); also occurs at low rates throughout life; resorption and formation are uncoupled and they occur on different surfaces; net bone growth

Agents that suppress REMODELING – fill in

resorption spaces

• Early in menopause, when baseline remodeling

rates are high  large BMD increases occur

• Senile osteoporosis, steroids, disease states –

low remodeling rates  minimal BMD increases

Baron and Hesse, JCEM, 2012

Agents that stimulate MODELING – grow new bone

wherever – considerable potential to rebuild skeletal mass (senile osteoporosis, low bone formation states, fracture non-unions, etc)

Why Do Bones Remodel?

Allows skeleton to --

Respond to mechanical loading (modeling) Repair microdamage (“wear and tear”) & prevent accumulation

• Maintains “quality control”

Microfracture Is Repaired through Targeted Remodeling

Segovia-Silvestre T et al, Hum Genet, 2009

Microfracture Is Repaired through Targeted Remodeling

Segovia-Silvestre T et al, Hum Genet, 2009 Glucocorticoids

Apoptosis

• f OB’s

& osteocytes Bisphosp, denosumab Inhibit or deplete

Why Do Bones Remodel?

Allows skeleton to --

• Respond to mechanical loading (modeling)
• Repair microdamage (“wear and tear”) & prevent

accumulation

• Maintains “quality control”

Release minerals (Ca and phosphate) & growth factors stored in matrix into circulation (& microenvironment)

• Important in skeletal homeostasis

Remodeling imbalances – important in aging and post-menopause

RANK-Ligand/ RANK/Osteoprotegrin Pathway

Osteoblasts & BM Stromal Cells Activated Osteoclast

TNF- PTH IL-1 PTHrP Glucocorticoids Vitamin D PGE2 IL-11

RANKL RANK Boyle WJ et al. Nature 2003;423:337; Hofbauer LC, Schoppet M. JAMA 2004;292:490

Osteoclastogenesis: Hormones, Growth Factors, Cytokines Stimulate Expression of RANK-L {RANK+RANK-L Interact}

IL-6

.

Pre-fusion Osteoclast CFU-M Multinucleated Osteoclast

+mCSF Bone Resorption

Osteoprotegerin (OPG) Prevents RANK- L/RANK Interaction & Inhibits OC Activity

[OPG=Circulating Inhibitor, product of OB cells]

Activated Osteoclast CFU-M Pre-fusion Osteoclast Multinucleated Osteoclast

Osteoblasts

Bone Formation Bone Resorption

Boyle WJ et al. Nature 2003;423:337 Hormones Growth Factors Cytokines

RANKL RANK OPG

X X X

Denosumab does the same thing

Baron R et al, Bone, 2011

Antiresorptive drugs act on osteoclasts with different mechanisms of action 

• Bisphos - OC’s stay on bone surface (drug – internalized,

long half-life)

• Denosumab – resorbing OC’s never form (MAb not

internalized – short half-life)

disrupt cytoskeleton arrest differentiation

Wnt/b-Catenin - Role in Bone Formation

OSTEOBLAST LINEAGE CELLS

Mesenchymal stem cells, pre-OB’s, mature OB’s, bone- lining cells, stromal cells, and osteocytes

• Produce matrix and mineralize it –

– Mechanical support – Matrix - reservoir of Ca, phosphate, growth factors, hormones – Secrete endocrine & paracrine factors – FGF23, DMP1, etc

• Modulate development of tri-lineages of blood cells
• Play role in metabolism, male reproduction

Function and numbers of cells in OB lineage decline with aging – many factors responsible

*

Wnt Signaling Pathway - 1

(Duan and Bonewald, Int J Biochem Cell Biol, 2016)

• Wnt/b-catenin pathway regulates cell fate throughout

lifespan

• Canonical Wnt pathway – depends on Wnt/b-catenin

signaling

• Non-canonical Wnt pathways – independent of b-

catenin (Ca2+, other signaling pathways)

• Many receptors, inhibitors, activators, modulators,

phosphatases, kinases, enzymes in Wnt pathway – complex regulation ฀ b-catenin – central molecule

• Key receptors – LRP4, 5, 6

Wnt Signaling Pathway - 2

(Duan and Bonewald, Int J Biochem Cell Biol, 2016)

• In bone and cartilage, Wnt important in:

– Skeletal development, limb patterning, chondrogenesis – OB differentiation/function/matrix synthesis – Differentiation and functioning of OC’s

• Targeted deletion of b-catenin in OB’s in mice 

– Reduced differentiation of MSC  OB and OB survival – Decreased [OPG], increased resorption – Low bone mass phenotype

• Targeted deletion of b-catenin in OC’s in mice

– Less clear-cut – Increased resorption

Wnt Signaling Pathway - 3

(Duan and Bonewald, Int J Biochem Cell Biol, 2016)

• Studies in osteocytes  Wnt pathway important

in sensing mechanical stimuli and responding to them

• Targeted deletion of b-catenin in osteocytes in

mice

– Progressive loss of bone mass with growth ( low BMD) – Increased osteoclast activity – due to less OPG

• Other pathways interact (cross-talk) with Wnt

signaling – estrogen, PTH, prostaglandin, BMP-2 (anabolic)

Canonical Wnt Signaling

Lewiecki et al, Nat Rev Rheumatol, 2011

NEW bone formation (quiescent & remodeling surfaces)

If no Wnt present, no signaling - β- catenin levels are LOW

• Wnt signaling (OB, OB

precursors)  recruits IC protein Axin which moves to tail of LRP5/6 (because of interaction with Dvl)

• Complex forms, recruits FRAT1

and glycogen synthase kinase-3 β

• Complex forms & inhibits β-

catenin phosphorylation

• Non-phos β-catenin

accumulates in cytosol, goes to nucleus

• β-catenin binds to LEF/TCF

elements and activates OB transcription program 

• RSPO & norrin modulate Wnt

Baron R, Kneissel M, Nat Med 2013

FINAL 

+ OPG

(osteoprotegerin)

• WIF1 (Wnt inhibitory

factor) or SFRP (secreted frizzled related protein) sequester Wnt ligand

• Axin & APC associate

with GSK-3 β increase phosphorylation of β- catenin

• β-catenin~P 

ubiquinated  proteasome for degradation

• NO bone made
• Other inhibitors: N-

cadherin inhibits LRP5/Wnt; sclerostin & DKK1

Baron R, Kneissel M, Nat Med 2013

Wnt Inhibition

 Bone

Pre-osteoblast lining cells Mature Osteoblasts Mesenchymal stem cells

Sclerostin*

Osteocyte

X X

New bone

Sclerostin Made by Osteocytes Negatively Regulates Bone Formation

Ott SM. JCEM 2005; Semenov M, et al. JBC 2005; Semenov MV, et al. JBC 2006; Li X, et al. JBC 2005

• Preclinical studies targeting gene (KO) and

transgenic overexpression of protein support these roles for Sclerostin

Clinical Studies: Sclerostin As Bone- Specific Molecular Target

• Sclerosteiosis – rare recessive genetic disorder of

high bone mass (bony overgrowth  CNS compression, death) – Heterozygous family members [1 functional sclerostin (Sost) allele] – healthy, have higher than normal BMD (high Z, high T scores - no

• ther complications) and normal life expectancy

– Reduced level sclerostin (lifetime)  tolerated

• Multiple clinical trials - Mab blocking sclerostin

(romosozumab) dose-dependently increases BMD and reduces fractures

Interaction of Bone and Fat: Physiologic and Disease States

Bone Marrow Fat

(Devlin M, Rosen CJ, Lancet Diab Endo, 2015)

• Bone and fat – juxtaposed in marrow cavities
• Dynamic fat depot
• Adipocytes release cytokines & adipokines
• Metabolic processes, hematopoiesis,
• steogenesis
• Osteoblasts and adipocytes come from the

same precursor  mesenchymal stem cell

• Bone and fat function as a ‘niche’
• BMAT - plays role in modulating
• steoporosis, aging, obesity, diabetes
• Niche/microenvironment
• Precursors   final cells – classical

view; likely to be ‘plasticity’ of cells along the way, phenotypic change

Devlin and Rosen, Lancet Diab, Endo 2015

Plasticity – pre-OB and pre- adipocytes

Communication

Bone lining cells  OB’s &

• steocytes

normally & adipocytes if injury

Bone Marrow Fat and BMD – Inverse Relationship*

(Devlin M, Rosen CJ, Lancet Diab Endo, 2015)

Bad for bone

*complex

Bone Marrow Fat

(Devlin M, Rosen CJ, Lancet Diab Endo, 2015)

• Function unclear; may be context-specific

(which bone)

• Fat found in all bones (15% of total fat =

BMAT)

• Two types of BMAT - -
• Constitutive (there all the time)
• Dynamic – regulated by local and systemic

factors

• Can’t distinguish anatomically or

functionally

BMAT- Diabetes, Osteoporosis

(Devlin M, Rosen CJ, Lancet Diab Endo, 2015)

• T1DM - high BMAT with impaired cortical

bone geometry (BMD – low, formation – low)

• T2DM – BMAT not high, but composition

different (more saturated lipid); BMD – ok, but fracture rate higher

• Premenopausal women with osteoporosis –

higher BMAT (vs controls)

• Aging cohorts: higher BMAT assoc with lower

trab BMD (qCT) in women; higher BMAT assoc with increased vertebral frx’s in men

Can you design a therapy to target BMAT and improve bone mass or quality?

PTH, PTH-1R and BMAT

(Fan Y et al, Cell Metab 2017)

• MICE - deleted PTH/PTHrP-1 receptor from

mesenchymal stem cells  low bone formation, high bone resorption, and high BMAT

• Separated/purified BM adipocytes from mice –

expressed high levels of RANK-ligand (!!)

• More RANK-ligand in serum and in bone marrow

supernatant

• PTH given intermittently to mice – DECREASES

BMAT

• Men with idiopathic osteoporosis – rec’ed PTH

(1-34) injections showed reduced BM fat by bone biopsies

Intermittent PTH and BMAT

Fan Y et al, Cell Metab, 2017

18 months PTH (1-34) treatment reduces BMAT (cell #)

Pathophysiology of Bone Loss

Menopause

• Remodeling increases, more BMU’s are formed,

deeper resorption pits (b)

• Amount of bone formed - less than what was resorbed
• Remodeling imbalance occurs (negative) -

“uncoupling”

• With time - structural deterioration of bone

– Thinned trabeculi, decreased connectivity, perforations

Lewiecki EM, Nat Rev Rheumatol, 2011

Effects of Estrogen on Bone Resorption

Stromal cells/Pre-OBs Monocytes Active

• steoclasts

E

OPG TGFb

IL6 mCSF IL11 GM-CSF RANKL

Early OC progenitors

resorption resorption

+E

• E

RANK +E

• E

IL1 TNF

+E +E M-CSF IL-6 IL-1 TNF RANKL

• Estrogen: dampens IL-1, TNFα  decreases IL6, IL11, GM-CSF, RANKL,

mCSF; increases OPG, TGF-β

• Estrogen deficiency: increases TNFα, IL1  releases IL6, M-CSF, IL11,

GM-CSF, RANKL  stimulates OC’s; decreases OPG, TGF-β

Khosla S, J Ger Med Sci, 2013

Mechanisms for Age-related Bone Loss -

• Sex steroid def present (women, men) + nutritional issues

(Ca & vitamin D def, often secondary HPT, sarcopenia)

• Intrinsic defects in marrow stromal cells with aging 

impaired proliferation & differentiation (“senescent OB’s”)

More fat

SUMMARY

• Skeletal homeostasis maintained by balance
• f resorption (RANK-L/RANK/OPG pathway)

and formation (Wnt/ b-catenin)

• Wnt pathway - bone formation (OB’s),

resorption (OC’s) and mechanotransduction (oc’s)

• Bone and fat (BMAT) – important influence
• n skeletal homeostasis
• Sex hormone deficiency and age-related
• steoporosis - imbalances in remodeling –

different mediators

• Current treatments target modeling and

remodeling

Targeting Bone Formation Therapeutically by Selective PTH/PTHrP Receptor Activation

Biased Agonism of Receptor - Different ligands favor formation of different PTH-R1 conformations (RG for PTHrP, R0 for PTH)

*Bone anabolic effects favored with strong peak responses but shorter

• verall cAMP

signals *Preclinical studies supported

*

Cheloha, RW et al. (2015) Nat. Rev Endo.

*

Comparison of Sequence Identity

Abaloparatide design: Potent bone anabolic activity with limited/no effects on resorption; analogue that favors binding to RG form of PTH-receptor (‘biased agonist’)

22 34

ABL hPTHrP hPTH

100% hPTHrP

38% hPTHrP

1

34

1

Novel analog of hPTHrP (1-34)

Abaloparatide

Abaloparatide Favors Binding to RG Form of PTH-R1 Abaloparatide Activates cAMP, Washes Away Fast

Hattersley et al, Endo 2015 ABL– low affinity ligand for R0

Targeted Remodeling - Complications

• Defects in microdamage repair could be important in

susceptibility to atypical femur fractures (BP-treated patients)

• Suggested by recent study –

– 3 sisters who had atypical femur fractures on oral bisphosphonate (2 unilateral, 1 bilateral) – Susceptibility looked genetic, likely a ‘dominant’ trait  whole exome sequencing on sisters and controls – All 3 sisters (+ 1 unrelated patient with an AFF) had same mutation in very highly conserved residue in geranylgeranyl pyrophosphate synthase (Asp188  Tyr)

– Projected to impair enzymatic function

Roca-Ayats N et al, NEJM, 2017

Mevalonate Pathway

• Mutation  impairs

GGPS enzyme activity

• BP’s block enzyme earlier

in the pathway

• Predicted to cause a

severe reduction in prenylated proteins

• Critical to OC function
• Reduced remodeling –

greater than predicted due to bisphos alone

• Could contribute to

susceptibility to AFF

• Reason – defective

microdamage repair/ adhesion/ruffled border

Gut Microbiome

(Steves et al, JBMR, 2015; Hernandez CJ et al, JBMR, 2016; Li et al, JCI, 2016)

• Gut MB  host metabolic pathways & innate & adaptive

immune systems Aging    Inflammation    Disease Microbiome

• Role in osteoporosis (+ sarcopenia, frailty)
• MODIFIABLE - - by probiotics (bacteria in food or dietary

supplements) and prebiotics (usu complex CHO fibers in fruits/vegetables)

• Potential to affect bone -
• Enhanced absorption of minerals
• Enhanced barrier function
• Enhanced immune system

Age-related Osteoporosis

• Imbalance in the bone formation response

to ongoing bone resorption

• Bone as tissue “ages”
• Changes in material properties – affect

strength – and in matrix components – affect constituents/composition - released into microenvironment (IGF-1, TGF-beta) Is the problem only with osteoblasts? Osteoclast lineage involved?

Factors Released from Bone with Osteoclastic Resorption

Sims NA, Ng KW, Curr Osteo Rep, 2014

• IGF-1
• TGF-β

– Promote bone cell proliferation, differentiation – TGF-β and IGF-1 levels in bone fall with age – Matrix changes with aging – May underlie reduced bone formation responses seen with aging in men and women

IGF-1 TGF-β IGF-1 TGF-β

Osteoclasts: Key Regulators of Bone Metabolism,

Release ‘Coupling Factors’, Act Directly on OB’s

 Process May Be Altered with Aging

TGF-b

OSTEOCLAST

TGF-b CXCL16 S1P Wnt 1

Pre- OSTEOCLAST Pre- OSTEOBLAST OSTEOBLAST

Wnts 1, 4,10b MSC

(provided by MJ Oursler)

Less TGF-b with age

Targeted Remodeling

• Defects in microdamage repair could be important in-

Fracture healing Susceptibility to atypical femur fractures (BP treated patients)

• Suggested by recent genetic study – 3 sisters with

atypical femur fractures on oral bisphosphonate

– Whole exome sequencing  3 sisters (+ 1 unrelated patient with an AFF) had same mutation in very highly conserved residue in geranylgeranyl pyrophosphate synthase (Asp188  Tyr)

– Projected to impair enzymatic function; affect OC function (ruffled border formation)

Roca-Ayats N et al, NEJM, 2017

Studies That Support Sclerostin As Target (to block)

• Pre-clinical:

– KO of sclerostin genes in mice produces high bone mass (Li et al, JBMR, 2008) – Treat rats with OVX-induced bone loss with anti- sclerostin MAb  increased trabecular and cortical BMD (Li et al, JBMR, 2009) – In rats with immobilization-induced bone loss, bone formation increased & resorption blocked with anti-sclerostin MAb (Tian et al, Bone, 2010) – In rat and monkey fracture models – healing improved with sclerostin MAb (good strength)

(Agholme et al, JBMR, 2010; Ominsky et al, JBMR, 2011)

DKK1 and Sclerostin (Bone)

• Scl and DKK1 bind Wnt

co-receptors LRP5/6 (block signaling)

• Use co-receptors to

augment inhibition

• DKK1 forms a ternary

complex (LRP5 or LRP6) and Kremen (receptor) 1

• r 2 - - this leads to

INTERNALIZATION of the complex

• Scl binds LRP4 –

enhancing its inhibition of Wnt

Ke et al, Endo Rev 2012; Baron & Kneissel, Nat Med 2013

Loss of Function Sclerostin Mutations

Both sclerostin genes mutated Child (A), adult (B,D) with SCLEROSTEOSIS

(C) Carrier with one mutant allele (1/2 dose)

Healthy adult with dense bone, elevated BMD T- and Z-scores (spine, hip) – no fractures or deformities (over lifetime)

Gardner JC et al, JCEM, 2005 D A B C

Coupling Factor Hypothesis (OCOB) - Osteoclast-Derived Factors

Wnt10b S1P BMP6 SCLEROSTIN

OSTEOCLAST PRE-OSTEOCLAST OSTEOBLAST PRE-OSTEOBLAST

Pederson et al. PNAS 105:20764, 2008

S1P/Rho GTPase Control of Osteoblast Lineage Cells

RhoA GTPase

Sphingosine-1 Phosphate

Migration Chemotaxis

OSTEOCLAST MSC/OSTEOBLAST

Quint et al, JBC 2013; (provided by MJ Oursler)

TGF-β Released From Bone Matrix During Resorption

TGF-b

OSTEOCLAST MSC Tang et al, Nat Med, 2009; (provided by MJ Oursler)

TGF-b

OSTEOBLAST

TGF-β from OC Activity – Influences Migration of OB Cells

Migration Sites of Resorption Fracture Repair

CXCL16 chemokine

TGF-b

Ota et al, Bone, 2013; (provided by MJ Oursler)

LIF Leukemia inhibitory factor

Osteoblasts

Osteoclasts Respond to TGF-β Released From Matrix During Resorption

TGF-b

OSTEOCLAST

TGF-b

(provided by MJ Oursler)

Amount with aging

Conditional Deletion of TGF-β Receptor II in Osteoclasts (mice)

(Weivoda et al, JBMR 31:76; 2016; provided by MJ Oursler)

RI RI RII

TGF-b

• Bone mass reduced  trabecular osteopenia
• Bone weaker by mechanical testing
• OB numbers, serum P1NP, bone formation

rates by histomorphometry (OB) - LOW Resembles senile bone loss OC’s  OB function (via Wnt)

Sham (control) Ovariectomy OVX + Sclerostin Ab

Li et al, JBMR, 2009

Micro-CT Images (rat) *

…& mechanically strong

• Estrogen present/therapy: dampens IL-1, TNF  decreases IL-6, IL-

11, GM-CSF, RANKL and mCSF; increased OPG

• Estrogen deficiency/menopause: INCREASED TNF-α, IL-1;

INCREASED release of IL-6, M-CSF, IL-11, GM-CSF, RANK-L  stimulate OC’s/OC activity; DECREASED OPG, TGF-β

(Tella, Gallagher, J Ster Biochem Mol Bio, 2014)

Sclerostin Inhibits Wnt Pathway *

New Bone Formation

Gennari L et al, Exp Opin Pharmacother, 2014

* Neutralizing MAb to sclerostin

Gut Microbiome

(Hernandez CJ et al, JBMR, 2016)

• Benefits to the host

– Vitamin production (many) – Extracts nutrients and energy from diet – “Metabolic function” (metabolites  host) – Regulates immune system – Protects against pathogens getting in

• How might the microbiome help bone?
• Enhance absorption of minerals (probiotics,

prebiotics)

• Enhance barrier function
• Enhance immune system (good or bad)

A LOT OF EVIDENCE FOR MB INVOLVEMENT FOR BONE IN HUMANS - MOST IS INDIRECT

Gut MB May Play a Fundamental Role in Bone Mass Regulation (Igbal et al, JCI, 2016)

Normal gut flora antigens (in MB) are presented to APC, T cells Pro-inflammatory cytokines made (ESTROGEN will normally dampen this, maintain barrier via gap junctions)

NO estrogen, these cytokines drive resorption systemically; barrier function also reduced (gap junctions – faulty)

Probiotics: Signal through APCs/T cells to reduce TNF-α, IL1-β, RANK- L; increase IL10 and OPG and Treg activity Increase TGF-β May be “estrogen-like” molecules  restore nl estrogen signaling and barrier function

bone mass

Sex Steroid Deficiency (SSD) Associated Bone Loss Is Microbiota-Dependent and Prevented by Probiotics (Li et al, J Clin Inv, 2016)

• Female mice (SSD)

–  gut permeability, expanded TH17 cells (OC-genic pop. T cells) –  OC-genic cytokines in small intestine, marrow (TNF, RANK-L, IL-17) – Bone loss (micro-CT, histomorphometry, BTM’s)

• These events don’t happen to mice kept under germ-free conditions.
• Twice weekly treatment of SSD mice with probiotic –

– Reduce/reverse trabecular bone boss (4 weeks after OVX) – No effects on cortical bone – Cytokines, T cell profiles (gut, bone) are ones that “less pro-resorptive”

• Probiotics improve trabecular BMD in control mice
• Several potential mechanisms postulated

*