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Function of C/EBP beta in bone development and bone tumourigenesis

Final Activity Report Summary - C/EBP beta and bone (Function of C/EBP beta in bone development and bone tumorigenesis)

Bone homeostasis is controlled by the interplay between cells that build up the bone, i.e. osteoblasts, and cells that degrade the bone, or osteoclasts. The dynamic processes of bone formation and resorption are tightly coupled, and disturbance of this balance causes several bone diseases. The differentiation, i.e. the process in which precursor cells develop into functional cells, as well as the function of both osteoblasts and osteoclasts are controlled by signalling molecules and proteins that regulate the expression of other proteins, the so-called transcription factors.

A transcription factor that is known to be important in the differentiation processes of several cell types is referred to as CCAAT / Enhaner binding protein beta (C/EBPß). C/EBPß was previously shown to be present in osteoblasts and suggested to have a function in their differentiation. In this project, we wanted to determine the precise function of C/EBPß in bone tissue, in terms of bone development and possible involvement in bone diseases. For this purpose, we analysed the bone tissue as well as the different bone cell types in genetically engineered mice that lacked C/EBPß. C/EBPß is normally present as three proteins differing in size that present functional parts in the protein. We additionally analysed mice which only expressed C/EBPß proteins, known as LIP, which lacked the ability to activate genes on their own.

Mice lacking C/EBPß showed a diminished bone mass. This decrease in bone mass was accompanied by the presence of smaller osteoblasts, which displayed a decreased function. Bone resorption was increased in these mice, because of the presence of enlarged osteoclasts with enhanced activity. This evidence demonstrated for the first time a function of C/EBPß in bone resorption and osteoclasts. The expression of only the LIP isoform restored the bone phenotype and even increased bone mass. Osteoblasts were larger and showed enhanced function, indicating that the presence of only this short isoform of C/EBPß was sufficient to support osteoblast differentiation. Similar to the C/EBPß-deficient mice, the LIP mice also demonstrated enhanced bone resorption caused by the presence of larger and more active osteoclasts. However, bone formation exceeded bone resorption, resulting in worse bone phenotype over time. Aged mice displayed an enormous increase in bone mass, even though the latter normally decreased in ageing animals. In some cases, this resulted in a bone marrow cavity that was completely filled up with bone mass and suggested that the LIP isoform was capable of transforming bone cells, being thus involved in bone pathological disorders.

The new finding of a function of C/EBPß in bone resorption and osteoclasts was further explored via culturing cells derived from the different engineered mice mutants and inducing these cells to differentiate into osteoclasts. Osteoclast cultures confirmed the formation of larger and more osteoclasts from cells originating from mice lacking C/EBPß or having only the LIP isoform present. These osteoclasts were functional and displayed normal behaviour. Surprisingly, osteoclasts were also formed from cells derived from the mutant mice, which were not induced to differentiate into osteoclasts. Under these conditions though, no osteoclasts developed in wild-type cultures. The cells from the mutant mice were shown to have an increased production of a signalling molecule known as TNFa, which was involved in many inflammatory processes. Blocking of this signalling molecule prevented the formation of osteoclasts in the non-differentiating conditions in mutant mice. In addition, blocking of TNFa also diminished the enhanced osteoclast formation in the osteoclast-inducing conditions.

It was therefore showed that C/EBPß was a key regulator of osteoclast differentiation and bone resorption, involving TNFa. In addition, C/EBPß was involved in both osteoblast and osteoclast functions, contributing to a correct bone homeostasis. Disturbance of C/EBPß resulted in an imbalance between osteoblasts and osteoclasts, rendering C/EBPß a possible novel therapeutic target in bone diseases.