Lehrstuhl für Orthopädie
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Die Enzyme TNSALP (Tissue Non-Specific Alkaline Phosphatase), ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) und ANKH (Ankylosis, progressive human homolog) bilden zusammen eine zentrale Regulierungseinheit für den Pyrophosphat (PPi)-Stoffwechsel der Zelle [1, 2].
Störungen dieses genau geregelten Prozesses resultieren in schwerwiegenden Erkrankungen, wie z.B. bei der Hypophosphatasie [3]. Dieser meist autosomal rezessiv vererbten Erkrankung liegt eine durch genetische Mutationen beeinträchtigte Funktion der TNSALP zugrunde, wodurch sich die PPi- Konzentration im Microenvironment der Zelle erhöht. Diese kann im Knochengewebe zu schweren Mineralisierungsstörungen führen [1, 2].
Andere Krankheiten, mit erniedrigten PPi- Konzentrationen, werden mit pathologischen Verkalkungen in verschiedensten Geweben in Verbindung gebracht [4, 5]. Diese gehen unter anderem auf genetische Defekte von ENPP1 zurück[4].
Auch der Mevalonat-Pathway trägt zur Komposition des Microenvironments bezüglich der Homöostase von Phosphaten bei [6, 7]. Hier bestehen auch medizinisch relevante Einflussmöglichkeiten, zum Beispiel durch Bisphosphonate, bei der sogenannten Volkskrankheit Osteoporose.
In dieser Arbeit wurden die Auswirkungen einer PPi-Belastung auf die in vitro Mineralisierung von Mesenchymalen Stammzellen untersucht, wobei Modulatoren der Enzymaktivität für ALP und ENPP1 und der Aktivität des PPi-Kanals ANKH sowie des Mevalonatstoffwechsels zum Einsatz kamen (PPi, Pyridoxalphosphat (PLP), Probenecid, Vitamin D, PPADS (Pyridoxalphosphat-6-azophenyl-2‘,4‘-disulfid Säure) und ß-γmeATP (ß-γ Methylentriphosphat)).
Die Resultate zeigen, dass die Modulation der PPi-Konzentration bei der osteogenen Differenzierung von hMSCs in vitro keine eindeutigen Effekte bewirkt. Geringe Änderungen des Genexpressionsmusters sind letztlich nicht auszuschliessen, blieben jedoch aufgrund der hohen Spendervariabilität durch eine erhöhte Anzahl von Experimenten zu beweisen.
Diese Arbeit zeigt insgesamt eine unerwartet geringe Auswirkung einer exogenen und endogenen Modulation der PPi-Konzentration sowohl mit Blick auf die rein physikalischen Phänomene der Mineralisierung, als auch mit Blick auf die untersuchte Genregulation der wichtigsten beteiligten Proteine, was möglicherweise die hohe Kompensationskapazität der Systeme unter physiologischen Bedingungen reflektiert. Untersuchungen auf proteomischer Ebene, besonders mit Blick auf die Prozessierung von Polypeptiden mit Mineralisierungs-modulierender Wirkung würden möglicherweise genaueren Einblick vermitteln.
Eine genauere Untersuchung der Einflüsse von ENPP1 erscheint für die Zukunft vielversprechend. Allerdings treten hier, besonders auch durch die verwendeten Hemmstoffe der ENPP1, die Phänomene der Vernetzung des Stoffwechsels der Phosphate (inklusive ATP und seiner Metabolite) mit dem Purinergen Signalling deutlich zutage. Diese Vernetzung generiert durch ihre Komplexität sowohl klinisch als auch zellbiologisch/biochemisch erhebliche Interpretationsprobleme, die zukünftige Arbeiten auflösen müssen. Dabei sollte besondere Aufmerksamkeit auf zwei für HPP-PatientInnen klinisch in Zukunft potentiell bedeutsame Ergebnisse gelegt werden, die möglicherweise ungünstigen Auswirkungen einer Therapie mit Probenecid auf die ALPL Expression und die Steigerung der ALPL Expression unter Hemmstoffen des Enzyms ENPP1.
1. Dympna Harmey, L.H., Sonoko Narisawa, Kirsten A. Johnson, Robert Terkeltaub, José Luis Millán, Concerted Regulation of Inorganic Pyrophosphate and osteopontin by Akp2, Enpp1 and Ank. American Journal of Pathology, 2003. 164, No. 4: p. 1199-1209.
2. Manisha C Yadav, A.M.S.S., Sonoko Narisawa, Carmen Huesa, Marc D McKee, Colin Farquharson, José Luis Millán, Loss of Skeletal Mineralization by the Simultaneous Ablation of PHOSPHO1 and Alkaline Phosphatase Function: A Unified Model of the Mechanisms od Initiation of Skeletal Calcification. Journal of Bone and Mineral Research, 2011. 26, No2: p. 286-297.
3. Beck, C., Hypophosphatasia. Klin Padiatr, 2009: p. 219-226.
4. Harmey, D.e.a., Concerted Regulation of Inorganic Pyrophosphate and Osteopontin by Akp2, Enpp1, and Ank. American Journal of Pathology, 2004. 164: p. 1199-1209.
5. Peter Nürnberg, H.T., David Chandler et all, Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia. Nature Genetics, May 2001. 28: p. 37-41.
6. Löffler, P., Heinrich, ed. Biochemie & Pathobiochemie. Vol. 8. 2007, Springer Verlag.
7. Joseph L. Goldstein, M.S.B., Regulation of the mevalonate Pathway. Nature Genetics, 1990. 343: p. 425-430.
Background
Hypophosphatasia (HPP) is a rare, inherited metabolic disorder caused by loss-of-function mutations in the ALPL gene that encodes the tissue-nonspecific alkaline phosphatase TNAP (ORPHA 436). Its clinical presentation is highly heterogeneous with a remarkably wide-ranging severity. HPP affects patients of all ages. In children HPP-related musculoskeletal symptoms may mimic rheumatologic conditions and diagnosis is often difficult and delayed. To improve the understanding of HPP in children and in order to shorten the diagnostic time span in the future we studied the natural history of the disease in our large cohort of pediatric patients. This single centre retrospective chart review included longitudinal data from 50 patients with HPP diagnosed and followed at the University Children's Hospital Wuerzburg, Germany over the last 25 years.
Results
The cohort comprises 4 (8%) perinatal, 17 (34%) infantile and 29 (58%) childhood onset HPP patients. Two patients were deceased at the time of data collection. Diagnosis was based on available characteristic clinical symptoms (in 88%), low alkaline phosphatase (AP) activity (in 96%), accumulating substrates of AP (in 58%) and X-ray findings (in 48%). Genetic analysis was performed in 48 patients (31 compound heterozygous, 15 heterozygous, 2 homozygous mutations per patient), allowing investigations on genotype-phenotype correlations. Based on anamnestic data, median age at first clinical symptoms was 3.5 months (min. 0, max. 107), while median time to diagnosis was 13 months (min. 0, max. 103). Common symptoms included: impairment of motor skills (78%), impairment of mineralization (72%), premature loss of teeth (64%), musculoskeletal pain and craniosynostosis (each 64%) and failure to thrive (62%). Up to now 20 patients started medical treatment with Asfotase alfa.
Conclusions
Reported findings support the clinical perception of HPP being a chronic multi-systemic disease with often delayed diagnosis. Our natural history information provides detailed insights into the prevalence of different symptoms, which can help to improve and shorten diagnostics and thereby lead to an optimised medical care, especially with promising therapeutic options such as enzyme-replacement-therapy with Asfotase alfa in mind.
The signal modelling framework JimenaE simulates dynamically Boolean networks. In contrast to SQUAD, there is systematic and not just heuristic calculation of all system states. These specific features are not present in CellNetAnalyzer and BoolNet. JimenaE is an expert extension of Jimena, with new optimized code, network conversion into different formats, rapid convergence both for system state calculation as well as for all three network centralities. It allows higher accuracy in determining network states and allows to dissect networks and identification of network control type and amount for each protein with high accuracy. Biological examples demonstrate this: (i) High plasticity of mesenchymal stromal cells for differentiation into chondrocytes, osteoblasts and adipocytes and differentiation-specific network control focusses on wnt-, TGF-beta and PPAR-gamma signaling. JimenaE allows to study individual proteins, removal or adding interactions (or autocrine loops) and accurately quantifies effects as well as number of system states. (ii) Dynamical modelling of cell–cell interactions of plant Arapidopsis thaliana against Pseudomonas syringae DC3000: We analyze for the first time the pathogen perspective and its interaction with the host. We next provide a detailed analysis on how plant hormonal regulation stimulates specific proteins and who and which protein has which type and amount of network control including a detailed heatmap of the A.thaliana response distinguishing between two states of the immune response. (iii) In an immune response network of dendritic cells confronted with Aspergillus fumigatus, JimenaE calculates now accurately the specific values for centralities and protein-specific network control including chemokine and pattern recognition receptors.
Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitously expressed enzyme that is best known for its role during mineralization processes in bones and skeleton. The enzyme metabolizes phosphate compounds like inorganic pyrophosphate and pyridoxal-5′-phosphate to provide, among others, inorganic phosphate for the mineralization and transportable vitamin B6 molecules. Patients with inherited loss of function mutations in the ALPL gene and consequently altered TNAP activity are suffering from the rare metabolic disease hypophosphatasia (HPP). This systemic disease is mainly characterized by impaired bone and dental mineralization but may also be accompanied by neurological symptoms, like anxiety disorders, seizures, and depression. HPP characteristically affects all ages and shows a wide range of clinical symptoms and disease severity, which results in the classification into different clinical subtypes. This review describes the molecular function of TNAP during the mineralization of bones and teeth, further discusses the current knowledge on the enzyme’s role in the nervous system and in sensory perception. An additional focus is set on the molecular role of TNAP in health and on functional observations reported in common laboratory vertebrate disease models, like rodents and zebrafish.