lecture 27

Question 1

Learning objectives?

Answer 1

• how is bone homeostasis maintained and why is it important to do so?
• what controls osteoclast differentiation?
• how do osteoclasts resorb bone?
• what regulates osteoblast differentiation?
• how do osteoblasts make bone?
• what are osteocytes?
• how do osteocytes control bone formation and bone resorption?
• why is wnt signalling important for bone?
• what regulates wnt signalling activation?
• what are some of the current treatments for osteoporosis and how do they work?

Question 2

What is the composition of bone?

Answer 2

outer cortical shell (thick, structural strength, protects inner bone and bone marrow)
cancellous/trabecular bone: struts that strengthen bone

organic matrix

• collagen = 90% of organic matrix and 10% of adult bone mass
• other proteins that help to bind mineral

mineral

• hydrosyapatite, an insoluble salt of calcium and phosphorous = 65% of adult bone mass
• small amounts of magnesium, sodium, and bicarbonate

water
- 25% of adult bone mass

Question 3

Is bone an idle tissue?

Answer 3

• no
• approx 5 - 10% of bone in an adult human skeleton is replaced each year
• this process is known as bone remodelling
• occurs at 1-2 million microscopic remodelling foci
• initiated asynchonously through the skeleton, at sites that are geographically and chronologically separated

Question 4

Why is our bone remodelled?

Answer 4

maintain mineral ion homeostasis

• 99% of body’s calcium, 85% of body’s phosphorous
• blood calcium maintained within a narrow range to maintain muscle and nerve function
• bone is resorbed to release calcium into blood when requried

adapt shape and structural organisation to alterations in biomechanical forces
- “mechanostat”

maintain structural integrity
- repair microdamage

Question 5

What is the BMU?

Answer 5

• basic multicellular unit
• the cellular makeup of the site of bone remodelling
• osteocytes: embedded within bone matrix, critical to maintaining bone health
• osteoblasts: bone forming
• osteoclasts: large, multinucleated cell, bone resorbing

Question 6

What is the physiology of bone remodelling?

Answer 6

• a balance in bone resorption and bone formation
• multinucleated osteoclasts come in and sit on bone surface
• resorb/eat a package of bone leaving resorption cavities (Lacunae)
• osteoblasts come in in teams releasing organic component of the bone
• unmineralised bone matrix = osteoid
• with time as a passive process osteoid binds minerals to become mineralised bone
• for bone to be healthy we need equal resorption and formation
• osteocytes do have a function in this process

Question 7

What is osteoid?

Answer 7

• unmineralised bone matrix

Question 8

How long does resorption/formation take?

Answer 8

• resorption takes about 3 weeks

- formation about 3-4 months

Question 9

What happens when we have increased osteoclast activity relative to osteoblast activity?

Answer 9

• net bone less e.g. osteoporosis
• thinner etc
• more susceptible to breaks

Question 10

What happens when we have more Obs relative to Ocls?

Answer 10

• net bone gain
• e.g. osteopetrosis, osteosclerosis
• genetic abnormalaties
• high bone mass
• more opaque in x-ray

Question 11

What is the osteoclast?

Answer 11

• the bone resorbing cells
• large multinucleated cells (4-20 nuclei/cell)
• enriched with mitrochondria

Question 12

How do osteoclasts resorb bone?

Answer 12

1. forming a “sealing zone” by integrin-mediated attachment to bone
2. acidification of resorptive lacunae to dissolve the mineral component
3. release of collagenases and other enzymes to degrade the organic component
4. bone degradation products are taken up by the osteoclast and released

bone resorption takes ~3 weeks

Question 13

What is the differentiation and function of osteoblasts?

Answer 13

• derived from mesenchymal/skeletal stem cell
• share a common cell fate with those to become muscle, chondrocytes
• osteogenic/adopogenic precursor: osteoblast or cells that produce fat tissue
• certain signals will make precursor cells follow osteoblast lineage
• in the mature osteoblast become bone forming cells that secrete bone matrix
• release some factors that aid bone mineralisation
• another function is support of osteoclast activity (early osteoblasts)
• early cells express a factor called RANKL
• later osteoblasts inhibit action of osteoclasts: OPG

Question 14

What are the three fates of osteoblasts?

Answer 14

• osteocytes
• lining cell (function unknown)
• apoptosis

Question 15

How do osteoblasts form bone?

Answer 15

Following cessation of osteoprogenitor proliferation:

• secretion of extracellular matrix proteins (osteoid) including collagen, bone, sialoprotein, osteocalcin
• expression of alkaline phosphatase
• -> renders the osteoid (non-mineralised matrix) competent for mineral deposition

Question 16

What regulates osteoclast differentiation?

Answer 16

• Receptor Activator of NF-KB Ligand (RANKL) and Osteoprotegrin (OPG) regulated osteoclast differentiation
• HSC –> myeloid precursor –> osteoblast precursor (expresses RANK) –> bound by sRANKL –> bone resorbing osteovlast
• OPG inhibits RANKL
  osteblast-lineage cells produce M-CSF (macrophage-colony stimulating factor (helps maintain osteoclasts))
  also produce RANKL
• mononuclear precursors fuse to form multinucleated cell once bound by RANKL
• only when they attach to a bone surface that they are a bona fide osteoclast
• multinucleated cells out in the middle of the bone marrow –> not osteoclast

osteocytes can also provide RANKL (somewhat controversial)

• if you take RANKL away osteoclast dies quickly

Question 17

What is RANKL?

Answer 17

also known as:

• ODF (osteoclast differentiation factor)
• trance (TNF-related activation-induced cytokine)
• OPGL) (osteoprotegrin ligand)
• member of TNF family of ligands
• mostley membrane-bound, but can be cleaved to a soluble form
• induces osteoclast differentiation and activation
• necessary and suffidcient for osteoclast generation
• RANKL -/- mice develop severe osteoporosis

Question 18

What is RANK?

Answer 18

• Receptor activator of NF-kB
• receptor for RANKL/ODF
• member of the TNF-receptor family
• expressed on osteoclasts and osteoclast precursors
• RANK -/- mice develop secere osteopetrosis
  i. e. there is no other factor in the body that will support osteoclast differentiation

Question 19

What is OPG?

Answer 19

• osteoprotegerin
• also known as: osteoclast inhibitory factor (OCIF)
• member of TNF-receptor family
• soluble receptor
• structurally distrinct from RANK but binds RANKL (deocy receptor)
• blocks RANKL/ODF-induced osteoclast differentiation and activation
• OPG -/- mice develop osteoporosis

Question 20

What ratio is critical to health of bone skeleton?

Answer 20

• RANKL:OPG
• key regulator of osteoclast differentiation
• increase in RANKL:OPG –> increase OCL –> net bone loss
• vice versa for OPG

Question 21

In summary how is osteoclast differentiation in healthy bone regulated?

Answer 21

• osteoclasts are derived from the myeloid cell lineage of haematopoietic stem cells
• M-CSF is required for proliferation of myeloid precursors and survival of osteoclast progenitors and mature osteoclasts
• RANKL is the essential cytokine required for osteoclast differentiation, survival and function
• • in healthy bone it is produced predominantly by early-mid stage osteoblasts; osteocytes may also produce RANKL
• RANKL binds RANK to signal to the cell to differentiate towards an osteoclast
• OPG is an endogenous decoy receptor for RANKL which prevents it from interacting with RANK, blocking osteoclast differentiation
• • in healthy bone mid-stage-mature osteoblasts produce OPG
• disruption of RANKL or RANK –> osteopetrosis due to absence of osteoclasts
• disruption of OPG –> osteoporosis due to proliferation of osteoclasts

Question 22

What promotes osteoblast differentiation and function?

Answer 22

Wnt ligands

• one example of a regulator of osteoblast differentiation
• act early on
• promote the proliferation of osteoblast precursors and their differentiation

wnt ligand binds Frizzled/LRP5/6 receptor

• results in stabilisation of signalling intermediate Beta-catening
• moves into nucleus to initiate gene transcription
• net positive effect of bone formation and net negative effect on bone resorption
• increase in expression of OPG

Question 23

In summary how does canonical Wnt signalling contribute to osteoblast and osteoclast differentiation and function?

Answer 23

• wnt ligands are soluble factors that act by binding the co-receptors Frizzled and LRP5 and LRP6
• this results in stabilisation of the signalling intermediate beta-catenin within the cytoplasm
• beta-catenin can then translocate to the cell nucleus where it can participate in activation of gene transcription
• • includes genes that have positive effect on osteoblast differentiation
• activation of WNt signalling can also inhibit osteoclast differentiation
• • beta-catenin can promote OPG expression, leading to increased OPG:RANKL, and inhibition of osteoclast differentiation

Question 24

What are endogenous antagonists of Wnt-ligand signalling in osteoblasts?

Answer 24

inhibits bone formation

• sclerostin
• DKK1 (dickkopf-1)
• bind LRP5/6 receptor
• secreted Frizzled related protein (sFRP1)
• binds Wnt ligand
• beta-catenin destablised and targeted for degradation
• ++ bone resorption
• – bone formation
• endogenous antagonists to the Wnt signalling pathway are made locally w/i hte bone microenvironment e.g. by osteoblasts
• DKK-1 (Dikkopf-1) and sclerostin inhibit canonical Wnt signalling by binding LRP5 or LRP-6 precenting these co-receptors from interactign with Frz, blocking activation of the Wnt signalling cascade (beta-catenin is destabilised and degraded)
• sFRPs (secered Frz related proteins) inhibit Wnt signalling by binding WNt ligands preventing them from binding the frz/LRP5/6/ co-receptor complex, blocking activation of the WNt signalling cascade
• absence of Wnt signalling activation leads to destabilistion and degradation of beta-catenin
• • this has a net negative effect (inhibitory effect) on osteoblast differentiation
• • can promote osteoclast differentation via increase in RANKL relative to OPG

Question 25

What is the osteocyte?

Answer 25

- ignored until last 5 - 10 years - terminally differentiated osteoblast, encased in bone matrix - can't just take them out of the body - most abundant bone cell, longest lived ~ average life span of 20-25 years - sit within the bone matrix in lacunae - have dendrite-like cell processes (canaliculi) that: - - enable intercellular communication between other osteocytes and to cells on the bone surface - - sense changes in mechanical loading - osteocytes regulate bone formation by changing expression of the Wnt signalling antagonists DKK-1 and sclerostin (inhibitors of bone formation)

Question 26

What is sclerostosis?

Answer 26

- patients have a mutation in sclerostin which renders it inactive - osteosclerotic condition, thick dense bone

Question 27

How are osteocytes mechanosensors of bone?

Answer 27

- increased mechanical loading - --> reduction in osteocyte expression of sclerostin and DKK-1 - promotion of bone formation, inhibition of bone resorption due to activation of Wnt signalling

Question 28

Are osteocytes are source of RANKL?

Answer 28

- maybe - if so they therefore influence osteoclast differentiation and survival - still controversial - not a key source

Question 29

What are treatment strategies targeting the osteoclast?

Answer 29

approved agents: - bisphosphonates - - zoledronate, ibandronate, alendronate, risedronate - - inhibits osteoclasts and many lead to osteoclast apoptosis - - act end stage, integrate into bone matrix and get taken up by osteoclasts anti-RANKL antibody - denosumab - fully humanised monoclonal Ab - inhibits osteoclast differentaiton and survival - expensive - both decrease in osteoclast number and function - - decreases fracture risk - - reduces bone loss - - no change in bone volume

Question 30

What are treatment strategies targeting osteoblast-lineage cells?

Answer 30

approve agent: - recombinant parathyroid hormone, amino acids 1-34 (rhPTH 1-34, teriparatide) - intermittent PTH (daily injection) is the ONLY APPROVED ANABOLIC THERAPY (increases bone mass) - promotes proliferation/maturation of precursors - downregulated sclerostin and DKK-1 emerging therapy: - anti-sclerostin mab - AMG785, romosozumab - phase II trial: post-menopausal osteoporosis - increased bone mineral density after 12 months compared to rhPTH 1-34 or placebo