Inhibition of signaling cascades in osteoblast differentiation and fibrosis

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Inhibition of signaling cascades in osteoblast differentiation and fibrosis

Krause, C.

Citation

Krause, C. (2011, October 5). Inhibition of signaling cascades in osteoblast differentiation and fibrosis. Retrieved from https://hdl.handle.net/1887/17892

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License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

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Modulating osteoblast differentiation – Concluding Remarks

Krause C.

2011

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Chapter 6 Modulating osteoblast differentiation – Concluding Remarks

The skeletal tissue is composed of various types of mesenchymal cells such as osteoblasts, chondrocytes, myoblasts and bone marrow stromal cells. These cells are believed to originate from common mesenchymal progenitors the so called pluripo- tent mesenchymal stem cells [7, 21, 13, 3].

Osteogenesis is a complex process that involves the differentiation of mesenchymal cells into pre-osteoblasts and osteoblasts that ultimately leads to the synthesis and deposition of bone matrix proteins (Figure 1) [18]. The regulation of osteoblast differentiation is mediated by local factors such as bone morphogenetic proteins (BMPs) and Wnt proteins (see chapter 1). Among this local factors, BMPs are the most potent regulators of osteoblast differentiation.They are believed to be the initiating factor of a myriad of signaling events that orchestrate bone formation [18] (see chapter 1-5).

Bone is continuously replacing itself by the action of bone resorbing osteoclasts and bone-forming osteoblasts, a process called bone remodeling. Because both cell types control each other’s activity there is a tight balance between these two pro- cesses. However, when the balance is disturbed by increased osteoclast or decreased osteoblast activity it can lead to disorders characterized by low bone mass such as osteoporosis.

A vast number of studies revealed that speciﬁc signaling systems through receptors and the speciﬁc inhibitors such as Noggin or Sclerostin modulate and coordinate BMP and/or Wnt functionality in bone remodeling (Figure 1).

6.1 Noggin – an inhibitor of bone formation

Like others, we have investigated the roles of BMPs involved in osteoblast differentiation and bone formation (chapter 2-3). Thereby, we could show in a comparative manner that different BMP proteins have varying osteogenic activity. In particular the difference of BMP-6 and its close paralog BMP-7 revealed a striking difference in bone anabolic activity. We therefore raised the question whether this difference in osteo- inductivity is caused by a differential interplay between BMP-6 and BMP-7 and the

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112 Chapter 6. Modulating osteoblast differentiation – Concluding Remarks

extracellular BMP antagonist Noggin. We observed that BMP-7 induces higher Noggin expression than BMP-6. Importantly, whereas BMP-7 was sensitive to Noggin inhibition, BMP-6 was not (Figure 1). We could further show that the mature domain of BMP-6, in particular a glutamic acid at position 60, confers Noggin resistance. This could lay the basis for an explanation why BMP-6 has a higher osteo-inductivity than BMP-7. But, if one takes in account that BMP-6 and BMP-7 have similar binding ca- pacities to Noggin, as we have shown via BIAcore measurements, one could speculate that these ligands transduce their signal differently. In particular when one takes in account that Noggin inhibits BMP-7 but not BMP-6 binding to overexpressed BMP receptors one could speculate that Noggin inhibition in the case of BMP-6:

• does really not exist

• follows a different signaling dynamic,

• occurs only in a different spatio-temporal setting,

• acquires facilitating receptors which are not present in the investigated cell type,

• the Noggin/BMP-6 complex binds a different subset of receptors then BMP-6 or even Noggin alone.

There are more and more evidences that BMP-6 exerts an eminent role in endodermal derived tissue maintenance. BMP-6 has been shown to be predominantly found in the liver, pancreas and the circulating system (see chapter 2). Thereby, BMP- 6 has been shown to be a key endogenous regulator of hepcidin expression and iron metabolism through the binding of hemojuvelin [1]. Furthermore, a role of BMP-6 as an reducer of glycemia via an insulin-independent pathway was just recently re- ported [12]. Whether Noggin is expressed in endodermal tissues and whether it exerts there any BMP(-6) antagonistic function is up to date not known (see chapter 2). Ad- ditionally, one has to take in account that antagonists might bind their own receptors, as it has been recently shown for the binding of gremlin to the VEGF receptor-2 [12].

Although we could not solve the Noggin/BMP-6 paradox, one needs to highlight that our ﬁndings facilitated the engineering of ’second generation’ BMPs with superior agonist activity with potential clinical applicability in spinal fusion, long bone non- union fracture treatment and osteoarthritis (see chapter 2-3).

6.2 Sclerostin – an inhibitor of bone formation

Although Sclerostin shares the inhibitory action on bone formation with the BMP antagonist Noggin, it is thought that the two proteins also have distinct activities. It is commonly believed that Sclerostin is speciﬁcally expressed by osteocytes and acts in a paracrine fashion on osteoblast derivatives (see chapter 4). Thereby, it exerts its

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6.2. Sclerostin – an inhibitor of bone formation 113

Mesenchymal stem cell

Pre – osteoblast

Mature osteoblast

Lining Cells

Osteocyte

Proliferation Maturation Termination

Osteo- chrondrogenic precursor

Immature osteoblast

Apoptosis

Sclerostin BMP7

BMP6

Sclerostin Noggin

?

Wnt3a, BMP7 Wnt signaling

Wnt3a

Figure 1: Schematic model summarizing the possible multiple inhibitory effects of Noggin and Sclerostin on the BMP and Wnt signaling pathways involved in osteoblast proliferation, differentiation and termination.

effects by opposing predominantly the action of the Wnt signaling pathway by shield- ing the binding site of Wnt proteins to the low density lipoprotein receptor-related protein (LRP)-4, -5, -6 [11, 5, 4, 9, 17].

Our experiments have reinforced the hypothesis that Sclerostin inhibits Wnt/β- catenin signaling but additionally we suggest that the inhibition by Sclerostin occurs in a paracrine and autocrine manner (see chapter 5). Whether Sclerostin exerts its effects by solely binding to LRP-5/-6 is questionable since our BIAcore data revealed an apparent low afﬁnity of Sclerostin for LRP-6. This leaves room for the assumption that

• the inhibitory action of Sclerostin is facilitated by a co-receptor

• Sclerostin may bind to its own, yet unknown receptor(s).

Whereas exogenous Sclerostin did not inhibit BMP signaling, our experiments revealed that Sclerostin can antagonize BMP/Smad signaling cell autonomously in the osteocytes (see chapter 5). When, Sclerostin and BMPs are co-expressed, Sclerostin was found to bind intracellular BMP-7. Thereby Sclerostin inhibits the secretion of BMP-7 and conveys its proteosomal degradation. Our BIAcore experiments revealed a similar low binding afﬁnity of Sclerostin to BMP-7 compared to the binding of Scle- rostin to LRP-6. Indeed, we could show that the binding of Sclerostin to BMP-7 is facilitated by binding of Sclerostin to the BMP-7 prodomain but one could also speculate that:

• the binding of Sclerostin to BMP-7 is supported by co-factors

• is facilitated through enrichment of Sclerostin and BMP-7 in ( secretory) vesi- cles.

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The physiological outcomes of inhibition of BMP/Smad signaling by Sclerostin was demonstrated by the increased BMP-7 signaling in in vivo SOST KO compared to wild type mice. Thereby, we could show for the first time that active BMP-7/Smad signaling is present in osteocytes of wild type mice which is further elevated in osteocytes of mice deficient of Sclerostin. One has to emphasize though that elevated BMP-7/Smad signaling in osteocytes of SOST KO mice was predominantly found at bone specific sites that are assumed to be in particularly load buffering, for example the calcaneus (heel bone). It is well known that osteocytes have mechanosensory properties, and mechanical loading triggers them to modulate bone homeostasis. In vivo, mechanical loading was found to reduce Sclerostin expression, thereby providing a mechanism by which bone formation upon mechanical loading is increased [14]. Therefore, one can speculate that the inhibitory action of Sclerostin on BMP-7 signaling in osteocytes is indirectly regulated through mechanical loading. Whether Sclerostin’s inhibitory action on BMP-7 signaling is reciprocally proportional to the applied mechanical load needs to be investigated in the future.

Osteocytes are described as key regulators of bone mass which regulate min- eral homeostasis through their apoptotic death and ultimate activation of osteoclasts.

That a link between Sclerostin and apoptotic death of osteocytes exist is commonly known [2, 6, 10, 16]. Consistent with this notion, we could show that in particular in areas of increased Wnt/β-catenin and BMP-7/Smad signaling of the distal tibia and the calcaneus, less apoptotic cells where found. Accordingly, we suggest that both Wnt/β- catenin and BMP-7/Smad signaling can act as pro-survival pathways for os- teoblasts and osteocytes which are both regulated by Sclerostin in order to maintain bone homeostasis. That Sclerostin acts directly on osteocytes, which then relays an- other inhibitory signal to osteoblasts can not be excluded from our experiments. A conditional BMP-7 targeted ablation in osteocytes as it has been done forβ-catenin would currently be the ultimate experiment to gain support of our hypothesis [8].

6.3 References

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6.3. References 115

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