Preventing mineralization found in pseudoxanthoma elasticum (PXE): the role of PPi

By Michelle Jaldin, Samar Rizvi,  Christine Vocke, Farrah Asgari-Majd, and Sharon Terry, PXE International
November 4, 2014

Lay Summary of Jansen RS, Küçükosmanoğlu A, de Haas M, Sapthu S, Otero JA, Hegman IEM, Bergen AAB, Gorgels TGMF, Borst P, van de Wetering K. ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release. Proc Natl Acad Sci USA. 2013 Dec 10; 110(50): 20206-11. PMID: 24277820

Note: Bold italicized terms are defined in the glossary

Pseudoxanthoma elasticum (PXE) is characterized by progressive ectopic mineralization of the skin, eyes and arteries. PXE is caused by mutations, or changes, in the ABCC6 gene.  This gene produces a protein, also called ABCC6.  Specifically, a person (or other organism) is affected by PXE when mutations inactivate or decrease the amount of the protein ABCC6.  In a mouse, inactivating the gene is called knocking out the gene, and the mouse is called a ‘knockout’ (KO) mouse.  ABCC6 KO mice develop PXE symptoms similar to those in humans and have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor, often called Factor X by researchers, in the circulation[1]. Conversely, when the ABCC6 gene is working effectively, and ABCC6 protein is present in the cells of the liver, then Factor X is transported out of the liver and into the circulation, where it prevents the abnormal mineralization of PXE. The identity of factor X and how it prevents PXE has been a mystery.

In a recent study funded by PXE International, a team of researchers in the Netherlands led by Drs. P. Borst and K. van de Wetering demonstrated that the factor, which normally prevents mineralization found in PXE, is inorganic pyrophosphate (PPi). In addition, they showed that PPi is not directly transported out of cells by ABCC6.  Instead, PPi is provided to the body’s circulation by supplying adenosine triphosphate (ATP), which is rapidly converted into adenosine monophosphate (AMP) and PPi by an enzyme known as ENPP1 (Figure 1).

Figure 1

Figure 1 - ATP equation



The steps taken to discover this are as follows:

1. ABCC6 protein prevents mineralization in cultured cells (in vitro).

First, researchers overexpressed the human and rat ABCC6 gene in HEK293 cells (human kidney cells). This means that they artificially increased the amount of the ABCC6 protein in the cells. They found that the solution, or medium, from cells with overexpressed human and rat ABCC6 interfered with mineralization, whereas medium from control cells (i.e. HEK293 cells with no over-expression) did not. These results show that the substance that prevents mineralization in vivo (in the body, in this case in mice) is also released in cultured cells, which contain ABCC6.

2. Identification of factors that prevent mineralization.

Their next step was to identify the ABCC6-dependent factor that prevents mineralization. First they searched with a technique called “metabolomics” for a compound that was less abundant in plasma of ABCC6 KO mice than in plasma of control (wild type [WT] or healthy) mice. Unexpectedly, they did not find any substance that was consistently more abundant in plasma of WT mice than in plasma of KO mice affected by PXE. Then the researchers used a similar approach to search in medium samples of HEK293-control and HEK293-ABCC6 cells for compounds that depended on the presence of ABCC6. Doing so, they found that the abundance of nucleosides, nucleoside monophosphates, and nucleotide sugars in the cell culture medium was highly dependent on the presence of ABCC6.  Of these, the most abundant ABCC6-dependent compound was AMP.

3. Nucleoside triphosphates are secreted by cells containing ABCC6.

Adenosine monophosphate (AMP) was exceptionally highly abundant in medium of HEK293-ABCC6 cells. However, AMP has no direct role in inhibition of mineralization. The researchers therefore hypothesized that the ABCC6-containing cells did not release AMP, but instead released adenosine triphosphate (ATP), which was rapidly converted into AMP and the mineralization inhibitor PPi after leaving the cells. The presence of PPi in the cell culture medium would also explain why medium of HEK293-ABCC6 cells inhibited mineralization. To provide evidence for this hypothesis they first tested whether medium of HEK293-ABCC6 cells contained PPi. These experiments revealed the presence of large amounts of PPi in medium of ABCC6-containing cells. Moreover, if inhibitors (ectonucleotidases) that prevent the conversion of ATP into AMP and PPi were added to the culture medium, this strongly reduced the ABCC6-dependent increase in PPi outside the cell and also resulted in an increase in ABCC6-dependent ATP. Similar results were obtained for other nucleoside triphosphates. These data suggest that nucleoside triphosphates are the predominant class of compounds secreted by cells containing ABCC6. 

4. PPi in KO mice is lower than in WT mice.

To test whether these results were of any relevance for living organisms (like mice or humans) they compared PPi levels in the plasma of ABCC6 KO (PXE) mice and WT (normal) mice, and found that the concentration of PPi was more than 60% lower in plasma from KO mice than in WT mice.


The authors of this study demonstrated that the factor that normally prevents PXE is inorganic pyrophosphate (PPi), which is known to inhibit mineralization of soft tissues. PPi is provided to the circulation in the form of nucleoside triphosphates (ATP) via a process that depends on ABCC6. The relationship of this cycle to PXE is supported by the in vivo evidence of decreased PPi levels in PXE KO mice compared to normal WT mice. These data suggest that PPi is the factor that normally prevents PXE. If reduced PPi levels cause mineralization, then PPi supplementation might be able to prevent it. However, PPi breaks down quickly in the circulation, and a suitable way to deliver it to the body is not yet available.


Additional Information

Jiang Q, Oldenburg R, Otsuru S, Grand-Pierre AE, Horwitz EM, Uitto J. Parabiotic Heterogenetic Pairing of Mice and Their Wild-Type Counterparts Halts Ectopic Mineralization in a Murine Model of Pseudoxanthoma Elasticum. The Am J Pathol. 2010 Apr;176(4), 1855-62. PMID: 20185580

Lay summary: Parabiotic mouse pairing experiments: more evidence that PXE is a metabolic disease.

Lay Summary: Clear evidence that PXE is a metabolic disease.



AMP: Adenosine monophosphate, a compound consisting of an adenosine molecule bonded to one acidic phosphate group, present in most DNA and RNA.

ATP:  Adenosine triphosphate, a compound used as an energy carrier in the cells of all known organisms.

Basolateral membrane:  A cell membrane that separates the inside of cells from the outside environment.

Ectopic mineralization:  Mineralization – deposition of calcium phosphate – in an abnormal place.

ENPP1: Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 is a compound that is encoded by the ENPP1 gene.  The compound participates in chemical reactions in the body that break specific bonds of nucleotides and nucleotide sugars, and release inorganic pyrophosphate (PPi). Mutations in the ENPP1 gene are associated with General Arterial Calcification of Infancy (GACI) and insulin resistance.

In vitro:  (Latin: in glass) Studies that are performed with cells studied outside their normal biological context.

In vivo:  (New Latin: in the living) Studies that test the effects of various biological treatments on whole, living organisms, usually animals, including humans.

KO mouse: A PXE KO - knock out - mouse is a genetically engineered mouse in which both copies of the ABCC6 gene are ‘knocked out´ so that they do not function, similar to a human with PXE.  A PXE KO mouse reliably develops a late onset of mineralization in elastic tissues that mimics PXE in humans.

Metabolomics:  The systematic study of metabolites, the substances produced in cells during metabolism, such as digestion or other chemical processes in the body.

Mutation:  Any change in a gene from its natural state.  The change may be disease causing or benign.  (Genetics Home Reference)

Nucleoside triphosphates: Molecules containing a nucleoside bound to three phosphates.  Nucleoside triphosphates are necessary for life, and have thousands of other roles in cell metabolism and regulation. (Wikipedia)

Orphan drug:  Orphan drugs are used in diseases or circumstances which occur so infrequently in the USA that there is no reasonable expectation that the cost of developing and making available in the USA a drug for such disease or condition will be recovered from sales in the USA for such drugs.  (orphanet)

WT mouse:  A WT – wild type - mouse is a mouse with no alterations in gene functions, so that all genes function normally.

[1] Jiang Q, Endo M, Dibra F, Wang K, Uitto J. Pseudoxanthoma elasticum is a metabolic disease. J Invest Dermatol. 2009 Feb;129(2):348-54. Free article in PubMed. PMID: 18685618