A bone mineralization defect in the Pahenu2 model of classical phenylketonuria involves compromised mesenchymal stem cell differentiation

Abstract

Osteopenia is observed in some patients affected by phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU). Bone density studies, in diverse PKU patient cohorts, have demonstrated bone disease is neither fully penetrant nor uniform in bone density loss. Biochemical assessment has generated a muddled perspective regarding mechanisms of the PKU bone phenotype where the participation of hyperphenylalaninemia remains unresolved. Osteopenia is realized in the Pah^enu2 mouse model of classical PKU; although, characterization is incomplete. We characterized the Pah^enu2 bone phenotype and assessed the effect of hyperphenylalaninemia on bone differentiation. Employing Pah^enu2 and control animals, cytology, static and dynamic histomorphometry, and biochemistry were applied to further characterize the bone phenotype. These investigations demonstrate Pah^enu2 bone density is decreased 33% relative to C57BL/6; bone volume/total volume was similarly decreased; trabecular thickness was unchanged while increased trabecular spacing was observed. Dynamic histomorphometry demonstrated a 25% decrease in mineral apposition. Biochemically, control and PKU animals have similar plasma cortisol, adrenocorticotropic hormone, and 25-hydroxyvitamin D. PKU animals show moderately increased plasma parathyroid hormone while plasma calcium and phosphate are reduced. These data are consistent with a mineralization defect. The effect of hyperphenylalaninemia on bone maturation was assessed in vitro employing bone-derived mesenchymal stem cells (MSCs) and their differentiation into bone. Using standard culture conditions, PAH deficient MSCs differentiate into bone as assessed by in situ alkaline phosphatase activity and mineral staining. However, PAH deficient MSCs cultured in 1200 μM PHE (metric defining classical PKU) show significantly reduced mineralization. These data are the first biological evidence demonstrating a negative impact of hyperphenylalaninemia upon bone maturation. In PAH deficient MSCs, expression of Col1A1 and Rankl are suppressed by hyperphenylalaninemia consistent with reduced bone formation and bone turnover. Osteopenia is intrinsic to PKU pathology in untreated Pah^enu2 animals and our data suggests PHE toxicity participates by inhibiting mineralization in the course of MSC bone differentiation.

Fig. 1.

Reduced Bone Formation in Pah^enu2. 1A. Hematoxylin and eosin staining of lumbar spine vertebrae four from wild type (left) and Pah^enu2 (right). H&E staining demonstrates Pah^enu2 has reduced bone mass, fewer trabecula and thinner trabecula compared to a wild type litter mate. 1B. Micro-CT reconstructed images of lumbar spine vertebrae four from wild type (left) and Pah^enu2 (right). Micro-CT demonstrates Pah^enu2 has reduced bone mass, fewer trabecula and thinner trabecula compared to a wild type litter mate. Micro-CT data and H&E data (1A) are in agreement. 1C. Static and dynamic histo-morphometry parameters. Panels A-C Static parameters A-C were generated from lumbar spine Micro-CT data of Pah^enu2 (six animals, 3 male, 3 female) and wild type litter mates (five animals, two male, three female). Micro-CT demonstrates in Pah^enu2 animals approximately 35% reduced mineral content, p = .04 (A) with increased trabeculae spacing, p = .05 (B) and increased bone volume/total volume, p = .025 (C). Panel D dynamic histomporphometry. Calcein labeling of Pah^enu2 (four animals, two male, two female) and wild type litter mates (four animals, two male, two female) demonstrates appositional bone growth was decreased 25%, p < .001.

Fig. 2.

Statistically significant biochemical metrics differing between Pah^enu2 and Controls. 2A. A moderate yet statistically significant reduction (p = .03) in plasma calcium is observed between PKU and control animals. 2B. A statistically significant reduction (p = .03) in plasma phosphate (p = .01) is observed between PKU and control animals. 2C. Moderate hyperparathyroidism is observed in Pah^enu2 animals (p = .02) indicating low Ca⁺⁺ and disruption of bone homeostasis. Cortisol, adrenocorticotropic hormone, and 25-hydro-xyvitamine D were assessed but no statistical differences in their representation were observed (data not shown). All measurements were made with four control animals (2 male, 2 female) and twelve PKU animals (5 male, 7 female).

Fig. 3.

Effect of hyperphenylalaninemia on Pah^enu2 and wild type MSC osteoblast differentiation. 3A. In situ alkaline phosphatase activity and von Kossa silver stain for mineralization in wild type control cells at standard culture conditions, wild type control cells in the context of 1200 μM PHE, PAH deficient cells at standard culture conditions, and PAH deficient cells in the context of 1200 μM PHE. Wild type cells (Pah replete) display abundant alkaline phosphatase activity and mineralization under standard conditions; however, mineralization is moderately reduced under hyperphenylalaninemic conditions. PAH deficient cells retain alkaline phosphatase activity and mineralization capacity under standard conditions; albeit reduced in comparison to PAH replete cells. PAH deficient cells under hyperphenylalaninemic conditions experience no loss of alkaline phosphatase activity while mineralization is reduced. 3B. Densitometry enables statistical analysis of alkaline phosphatase activity and mineralization. Alkaline phosphatase activity in PAH replete cells is not influenced by hyperphenylalaninemia. In PAH deficient cells alkaline phosphatase activity is significantly reduced compared to PAH replete cells (P < .01) but hyperphenylalaninemia does not cause further reduction of alkaline phosphatase activity in PAH deficient cells. PAH replete cells generate more robust mineralization than PAH deficient cells (P < .001). Hyperphenylalaninemia causes modest yet significant down-regulation of mineralization in PAH replete cells (P < .01) while in PAH deficient cells, hyperphenylalaninemia has greater impact to down-regulate mineralization (p < .001).

Fig. 4.

Effect of hyperphenylalaninemia on expression of Col1A1 and Rankl during Pah^enu2 and wild type MSC osteoblast differentiation (A-B). Expression data is calculated relative to Gapdh. All expression studies were performed in triplicate. 4A. Expression of Collai in wild type cells (Pah replete) is abundant during osteoblast differentiation reflecting robust bone formation. Wild type cells under hyperphenylalaninemic conditions display lower Collai expression. Pah deficient cells express Collai at levels similarly to wild type cells under hyperphenylalaninemic conditions reflecting residual bone forming capacity. Pah deficient cells under hyperphenylalaninemic conditions display the lowest Collai expression. 4B. Expression of Rankl in wild type cells (Pah replete) is abundant as greater bone turn-over occurs as a consequence of high level bone formation. Under hyperphenylalaninemic conditions wild type cells express less Rankl as lower bone turn-over will occur owing to lower bone formation. In Pah deficient cells under standard culture conditions Rankl expression is reduced owing to lower bone formation with subsequent lower bone turn-over. Pah deficient cells under hyperphenylalaninemic conditions form little bone, exceedingly little Rankl expression is observed as bone turn-over is not required.