Wednesday, December 25, 2013

Why do chondrocytes stop in achondroplasia?


The purpose of this article is to revisit the origin of achondroplasia from a new perspective, inspired by a very interesting article from Dr. Pavel Krejci, an enthusiastic researcher of fibroblast growth factor receptor 3 (FGFR3) biology and a strong contributor for the research in achondroplasia, published just few weeks ago.(1) The text here tries to explain the hypothesis brought by Dr Krejci.

This short review will also helps us to get into the next article planned for the blog, which is about blocking the enzymatic cascade discussed here. I know that the 17 original blog readers may find this text a bit repetitive but, while we gain more insight about achondroplasia, there is always a piece more of detail included. 

Life is electric 

Life runs on electricity. Every single moment of a living being is ruled by coordinated chemical reactions conducting electric charge transfers. If we take an instant to remember that in the body there are thousands and thousands molecules participating in this coordinated chemical program, we can start to see how life is complex. Can you imagine the kind of "software" needed to control this amazing machine? A chief program to run the life machinery does need to be very robust and strong. 

However, at the same time life is run by an incredibly strong program, it is also very fragile. With billions of chemical (electric) reactions occurring in the body in just one day, errors are prone to arise. It needs just one uncharted mistake in building a new molecule inside the machine and the balance could be broken, leading the body from a healthy state to disease. A bug in the software and consequences could be severe to the machine. 

Notwithstanding, the machine has its own quality control software running together with the chief one, so when things go wrong, the guardian software is activated and most errors are corrected very fast. In a cell, if the quality control software detects an error and can't correct it, other control measures start, and the cell can drive itself to death or to a paralyzed state called cell senescence (2,3) (save this for later).

Proteins, electric switches

In the body, the masters of those chemical (electric) reactions driving life are molecules we call proteins. Proteins are large structures made of smaller ones, called amino acids. Although many amino acids exist in nature, there is a limited number of them considered essential for life. All proteins in the body are made of the same amino acids and it is their specific combinations, written in the DNA code, that will make different functional proteins. Amino acid combinations are key because each one has its own electric properties. So, you see here, if a protein conduct electric reactions in a certain way due to its conformation, then imagine what can happen when its conformation is wrong. An amin acid in the wrong position and the protein may become inactive or too active.

Although being key rulers of life, not all proteins conduct chemical reactions and when they do so, they are called enzymes. We can think in enzymes like chemical (electric) wall switches. Their functions allow the body to work correctly and to react very fast to internal and external stimuli.

Figure 1. Proteins are like electric switches

Manutenção & Suprimentos 
Usually, enzymes don’t work alone. On the contrary, most of the time they act in concert with others in a way that we could compare them with blocks of a domino cascade, where the first block hits another and this one hits a third, to generate an effect in the end of the row. In the case of enzymes, the chemical reaction, or signal, will make the cell which is receiving it to behave in an appropriate way to respond to the cause of the signal.

Figure 2. An enzyme cascade is like a domino chain
A chemical signal may start as a response to the weather outside, at a ski resort or at the beach. Either way, cold or hot weather, the body will respond to balance the internal temperature with that of the environment, through hundreds of chemical reactions across the tissues and organs. As mentioned above, if all signals are under control, the body will respond in a proper way, otherwise, there will be problem ahead.

Understanding achondroplasia

All of these concepts are important to review because they can help us to learn what happens in clinical conditions such as achondroplasia. In achondroplasia, one of those active proteins, FGFR3 (Figure 3), contains a modification in its structure, a mutation, that makes it more active than usual. If the building mistake happened in an adult body, it is likely that the quality control software would pick it and solve it, otherwise the error could drive to cancer (see below). 

However, as in achondroplasia the mistake is built in (what is called in technical jargon a germline mutation), the mutation is not recognized as a mistake (it is inside the DNA code from the beginning) and the quality control will not recognize it as an error. If an error is causing so much trouble from the beginning, the new form of life in development (the embryo) would not be successful. In achondroplasia, the mistaken FGFR3 exerts an undue influence from the beginning (it impairs bone growth already in utero), but keeps being compatible with life. Nevertheless, with a workaholic receptor enzyme in the machine, the development program will have trouble (see below).

Figure 3. A structural model of FGFR3

Chemical signals driving achondroplasia 

After birth, FGFR3 is almost exclusively produced, that is, in a relevant way, by cells called chondrocytes, living within a very specialized region in the extremities of the long bones, the cartilage growth plate. The normal function of FGFR3 is to help chondrocytes to control their own proliferation (multiplication) and maturation speed. If FGFR3 is absent, bones grow excessively and ill consequences also arise. (4) Therefore, in normal conditions, FGFR3 is an important natural growth brake for chondrocytes. However, since in achondroplasia FGFR3 is signaling in excess, the result is that chondrocytes simply stop to proliferate and mature, leading to a general bone growth arrest and to the clinical features of this chondrodysplasia.

We mentioned that enzymes do not work alone, and FGFR3 is not an exception. It relies on a number of enzyme cascades to exert its functions (Figure 4) and we are going to revisit one of them, the mitogen-activated protein kinase (MAPK), which was the one explored by the study we will see in the next article.

The MAPK pathway

Over the last years, an increasing amount of evidence has been published pointing the MAPK enzymatic cascade as the likely key driver of the bone growth arrest seen in achondroplasia. (5)

Figure 4. FGFR3 signaling cascades

For instance, several studies exploring the use of c-type natriuretic peptide (CNP) have contributed much for the understanding of how MAPK signaling impairs bone growth. (6-9) This in turn resulted in the development of the first potential pharmacological therapy for achondroplasia, the CNP analogue BMN-111 (10), now about to enter phase 2 of its clinical development. To learn more about the clinical development of a new potential drug you could visit this previous article of the blog.

FGFR3 is a chemical antenna
The MAPK cascade is represented in the above figure 4 in a very simplified way. In short, FGFR3 is called a receptor enzyme (or technically, a receptor tyrosine kinase) because it is placed across the cell membrane (the cover) of the chondrocyte (Figure 3), like an antenna on the roof of a building, waiting for TV signals coming from outside. The natural state of FGFR3 is inactive (or power off). We have already reviewed this topic in other articles of this blog. 

Figure 5. FGFR3, a cell roof antenna

Wikimedia commons
FGFR3, like a TV antenna ready to receive only TV waves, will receive signals only from some specific proteins called FGFs. When a FGF binds FGFR3, it turns the receptor active (or power on), causing a change in the body of FGFR3 that will generate an electric charge, the signal, which is produced by the dislocation of some phosphorus ions (a phenomenon called phosphorylation) in the part of the receptor placed inside the cell. 

This electric / chemical signal will be driven to other molecules nearby and, by travelling from one to the next, like in the domino chain, the signal will reach the first enzyme of the MAPK cascade, which is called Ras. Ras is turned on and will activate the next enzyme in the pathway, called Raf, which will then activate MEK, which in turn will transfer the signal to another enzyme called ERK. ERK is the messenger that will take the signal brougth by the FGF upstairs to the cell nucleus, where the cell program will react accordingly (Figure 3). I suggest you to watch the following 14 min video, showing a rich and detailed animation of a signaling cascade in action, with the corresponding cell response. Cells can respond to external signals by producing new proteins and the animation shows this process. 

DNA Learning Center by Cold Spring Harbor Laboratory

The MAPK cascade signaling in chondrocytes

The MAPK pathway is a key driver of important cell functions in all tissues of the body. This implies that MAPK responds to a large number of cell antennas, not only FGFR3 (visit this article in Wikipedia to see the figure showing known MAPK interactions). 

Normally, MAPK works as a growth trigger: cells estimulated via MAPK start to multiply and survive longer. This property is readily recognized by cancer cells, which frequently use MAPK to grow more and fast. It is not surprising that lately, we have been witnessing the development of several new compounds targeting enzymes of the MAPK cascade to treat several kinds of cancer (reviewed here).

However, as we have just mentioned above, chondrocytes react to MAPK exactly in the opposite way, and reduce their growth pace under the influence of MAPK signaling. (11) How can this happen? Dr. Pavel Krejci's hypothesis might have answered this question. (1)

In his review, he postulates that this chondrocyte behavior would be a natural reaction to what they recognize as a wrong signal coming from the mutated FGFR3. In other words, due to the over active FGFR3 producing an excess of growth stimulation in chondrocytes of the growth plate, these cells defend themselves by entering in a kind of stop mode, the above mentioned cell senescence. They switch off the electricity.

Do you remember what we mentioned in the beginning of this article, that there is a quality control program running together with the master program which runs the body machine, to ensure that the chemical reactions are going in the right way? So, the hypothesis by Dr Krejci may be a good explanation for the exceptional behavior of chondrocytes facing an over active FGFR3. The cell might not see the mutated FGFR3 as an issue, but can perceive something is going wrong with the excessive MAPK signaling. 

Therefore, achondroplasia would be the final consequence of chondrocytes "diagnosing" the excessive signaling of MAPK as a problem, thus activating the quality control software to enter in a kind of stop mode, to protect themselves from the potential harm of excessive growth stimulation which we know is common in cancer. They keep with basic cell functions, but stop the proliferation program essential for bone growth. The result is shorter long bones, narrow spine canal and the clinical consequences seen in achondroplasia.

FGFR3 and MAPK in bone cells

Since there is evidence that the overactive FGFR3 also causes change in bone cells (osteoblasts), it is also useful to see what happens with those cells under influence of the FGFR-MAPK pathway. We do have information about this, for instance coming from a study by the Dr Sunichi Murakami's group. In one of their recent works, they explored the inhibition of FGFR3 and MEK in osteoblasts in vitro and in vivo, and concluded that the MAPK pathway is also very important for bone health. (12)


One question we can ask about the hypothesis by Dr. Krejci is related to the actual mechanism of action of FGFR3. If FGFR3 is a natural brake for bone growth, and acts naturally through the MAPK cascade, why would
chondrocytes launch the senescence process, since the essential signal ("stop growing") has not changed? Perhaps what activates the senescence process is an internal "cut-off" of the MAPK pathway activity, which when crossed, triggers the process of growth pause, but the mechanism by which this would be happening is unclear.

Taking advantage of the discussion on the MAPK cascade actions, i
n the next article we will review another study, where researchers tested a couple of small molecules created to target the MAPK pathway in cancer. What is relevant in their work is that they explored those compounds in bone and in the growth plate, with consequent implications for several strategies in development to treat achondroplasia.


1. Krejci P. The paradox of FGFR3 signaling in skeletal dysplasia: why chondrocytes growth arrest while other cells over proliferate. Mutat Res Rev Mutat Res. 2014;759:40-8.

2. Campisi J & Fagagna FA. Cellular senescence: when bad things happen to good cells. Nature Rev Mol Cell Biol 2007;8:729-40.

3. Campisi JCell biology: the beginning of the end. Nature 2013; doi:10.1038/nature12844. Published online 18 December 2013.

4. Toydemir RM et al. Novel mutation in FGFR3 causes camptodactyly, tall stature, and hearing loss (CATSHL) Syndrome. Am J Hum Genet 2006;79(5):935-41. Free access

5. Foldynova-Trantirkova S et al. Sixteen years and counting: the current understanding of 
fibroblast growth factor receptor 3 (FGFR3) signaling in skeletal dysplasias. Hum Mutat 2012; 33:29–41. Free access

6. Krejci P et al. Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis. J Cell Sci 2006; 118: 5089-00.

7. Nakao K et al. Impact of local CNP/GC-B system in growth plates on endochondral bone growth. Pharmacol Toxicol 2013; 14 (Suppl 1):48.

8. Yasoda A et al. Systemic Administration of C-Type Natriuretic Peptide as a Novel Therapeutic Strategy for Skeletal Dysplasias. Endocrinology 2009;150: 3138–44.

9. Yasoda A and Nakao K. Translational research of C-type natriuretic peptide (CNP) into skeletal dysplasias. Endocrine J 2010; 57 (8): 659- 66.

10. Lorget F et al. Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia. Am J Hum Genet 2012;91(6):1108-14. Free access

11. Murakami S et al. Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes Dev 2004;18(3):290-305. Free access

12. Kyono A et al. FGF and ERK signaling coordinately regulate mineralization-related genes and play essential roles in osteocyte differentiation. J Bone Miner Metab 2012;30(1):19-30. Free access

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