René St-Arnaud

Targeted Inactivation of the 25-Hydroxyvitamin D3-1α-Hydroxylase Gene (CYP27B1) Creates an Animal Model of Pseudovitamin D-Deficiency Rickets1

Pseudovitamin D-deficiency rickets is caused by mutations in the cytochrome P450 enzyme, 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase). Patients with the disease exhibit growth retardation, rickets, and osteomalacia. Serum biochemistry is characterized by hypocalcemia, secondary hyperparathyroidism, and undetectable levels of 1alpha,25-dihydroxyvitamin D(3). We have inactivated the 1alpha-OHase gene in mice after homologous recombination in embryonic stem cells. Serum analysis of homozygous mutant animals confirmed that they were hypocalcemic, hypophosphatemic, hyperparathyroidic, and that they had undetectable 1alpha,25-dihydroxyvitamin D(3). Histological analysis of the bones from 3-week-old mutant animals confirmed the evidence of rickets. At the age of 8 weeks, femurs from 1alpha-OHase-ablated mice present a severe disorganization in the architecture of the growth plate and marked osteomalacia. These results show that we have successfully inactivated the 1alpha-OHase gene in mice and established a valid animal model of pseudovitamin D-deficiency rickets.

Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process

Fibroblast growth factor 23 null mice (Fgf‐23−/−) have a short lifespan and show numerous biochemical and morphological features consistent with premature aging‐like phenotypes, including kyphosis, severe muscle wasting, hypogonadism, osteopenia, emphysema, uncoordinated movement, T cell dysregulation, and atrophy of the intestinal villi, skin, thymus, and spleen. Furthermore, increased vitamin D activities in homozygous mutants are associated with severe atherosclerosis and widespread soft tissue calcifications; ablation of vitamin D activity from Fgf‐23−/− mice, by genetically deleting the 1α(OH)ase gene, eliminates atherosclerosis and ectopic calcifications and significantly rescues premature aging‐like features of Fgf‐23−/− mice, resulting in prolonged survival of Fgf‐23−/−/1α(OH)ase−/− double mutants. Our results indicate a novel role of Fgf‐23 in developing premature aging‐like features through regulating vitamin D homeostasis. Finally, our data support a new model of interactions among Fgf‐23, vitamin D, and klotho, a gene described as being associated with premature aging process.

The 25-Hydroxyvitamin D 1-Alpha-Hydroxylase Gene Maps to the Pseudovitamin D-Deficiency Rickets (PDDR) Disease Locus

Pseudovitamin D‐deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25‐hydroxyvitamin D‐1α‐hydroxylase, a renal cytochrome P450 enzyme (P4501α) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13‐q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P4501α gene (hereafter referred to as 1α‐OHase). We have used a probe derived from the rat 25‐hydroxyvitamin D‐24‐hydroxylase (CYP24; 24‐OHase) sequence to identify and clone the 1α‐OHase cDNA. The full‐length 1α‐OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time‐of‐flight mass spectrometry. The expression of the gene was analyzed in vitamin D–replete animals; treatment with 1α,25(OH)2D3 reduced 1α‐OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2‐fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1α‐OHase gene mapped to 12q13.1‐q13.3, providing strong evidence that a mutation in the 1α‐OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1α‐OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.

Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes

Chondrocyte proliferation and differentiation requires their attachment to the collagen type II–rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

Podocyte-specific deletion of integrin-linked kinase results in severe glomerular basement membrane alterations and progressive glomerulosclerosis.

Alterations in glomerular podocyte cell-cell and cell-matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell-matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, alpha3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function.

The direct role of vitamin D on bone homeostasis

Vitamin D requires two metabolic conversions, 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney, before its hormonal form, 1,25-dihydroxyvitamin D [1,25-(OH)2D], can bind to the vitamin D receptor (VDR) to modulate gene transcription and regulate mineral ion homeostasis. The receptor and metabolic enzymes are expressed in many tissues, however, which has long suggested that the vitamin D hormone could act in an autocrine, paracrine, or intracrine fashion to affect the biology of non-classical target tissues. Strong support for this model has been obtained for Toll-like receptor-mediated innate immunity in macrophages, for example. The classical view is that vitamin D exerts its effects on bone indirectly via control of calcium and phosphate homeostasis, despite expression of cyp27b1, the 25-hydroxyvitamin D-1alpha-hydroxylase, and the VDR in osteoblasts and chondrocytes. Recent molecular genetic studies have revealed direct, but non-essential roles for 1,25-(OH)2D in growth plate chondrocytes. Specific inactivation of the VDR in collagen type II-expressing chondrocytes leads to reduced RANKL expression and delayed osteoclastogenesis, which causes a transient increase in bone volume at the primary spongiosa. Chondrocyte-specific VDR-ablated mice also show reduced circulating levels of FGF23 and thus elevated serum phosphate concentrations. The mechanisms remain to be completely determined but appear to involve a 1,25-(OH)2D-induced secreted factor from chondrocytes that affects FGF23 production by neighboring osteoblasts. The phenotype of additional mutant mice models, including chondrocyte-specific inactivation or overexpression of cyp27b1, is being analyzed to provide further support for these results that show autocrine and paracrine roles for 1,25-(OH)2D during endochondral bone development.

Integrin-linked kinase regulates endothelial cell survival and vascular development

ABSTRACT Integrin-linked kinase (ILK) is a phosphoinositide 3-kinase-dependent serine/threonine kinase that interacts with β integrins. Here we show that endothelial cell (EC)-specific deletion of ILK in mice confers placental insufficiency with decreased labyrinthine vascularization, yielding no viable offspring. Deletion of ILK in zebra fish using antisense morpholino oligonucleotides results in marked patterning abnormalities of the vasculature and is similarly lethal. To dissect potential mechanisms responsible for these phenotypes, we performed ex vivo deletion of ILK from purified EC of adult mice. We observed downregulation of the active-conformation of β1 integrins with a striking increase in EC apoptosis associated with activation of caspase 9. There was also reduced phosphorylation of the ILK kinase substrate, Akt. However, phenotypic rescue of ILK-deficient EC by wild-type ILK, but not by a constitutively active mutant of Akt, suggests regulation of EC survival by ILK in an Akt-independent manner. Thus, endothelial ILK plays a critical role in vascular development through integrin-matrix interactions and EC survival. These data have important implications for both physiological and pathological angiogenesis.

Correction of the abnormal mineral ion homeostasis with a high-calcium, high-phosphorus, high-lactose diet rescues the PDDR phenotype of mice deficient for the 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1)

Mutations in the 25-hydroxyvitamin D-1alpha-hydroxylase gene (CYP27B1; 1alpha-OHase) cause pseudo vitamin D deficiency rickets (PDDR), while mutations in the vitamin D receptor (VDR) cause hereditary vitamin D resistance rickets. Animal models of both diseases have been engineered. The bone phenotype of VDR-ablated mice can be completely rescued by feeding the animals with a high-calcium, high-phosphorus, high-lactose diet. We have attempted to rescue the PDDR phenotype of mice deficient for the 1alpha-OHase gene by feeding them with the high-calcium diet. The rescue regimen consisted of feeding a diet containing 2% calcium, 1.25% phosphorus, 20% lactose (rescue diet) from 3 weeks of age until sacrifice at 8.5 weeks of age. Blood biochemistry analysis revealed that the rescue diet corrected the hypocalcemia and secondary hyperparathyroidism. Despite the restoration of normocalcemia, 1alpha-OHase(-/-) (and 1alpha-OHase(+/-)) animals fed the rescue diet initially gained weight less rapidly than control mice fed normal mouse chow. Although 1alpha-OHase(-/-) mice fed the rescue diet eventually reached the same weight as control animals, the treatment did not entirely correct bone growth, as femur size remained significantly smaller than that of control. Bone histology and histomorphometry confirmed that the rickets and osteomalacia were cured. The rescue diet also restored the biomechanical properties of the bone tissue within normal parameters. These results demonstrate that correction of the abnormal mineral ion homeostasis by feeding with a high-calcium rescue diet is effective to rescue the PDDR phenotype of 1alpha-OHase mutant mice. This treatment, however, does not appear as effective as 1,25(OH)(2)D(3) replacement therapy since bone growth remained impaired.

Integrin-Linked Kinase Deletion from Mouse Cortex Results in Cortical Lamination Defects Resembling Cobblestone Lissencephaly

Integrin-linked kinase (Ilk) is a scaffold and kinase that links integrin receptors to the actin cytoskeleton and to signaling pathways involved in cell adhesion, migration, and extracellular matrix deposition. Targeted deletion of Ilk from embryonic mouse dorsal forebrain neuroepithelium results in severe cortical lamination defects resembling cobblestone (type II) lissencephaly. Defects in adult mutants include neuronal invasion of the marginal zone, downward displacement of marginal zone components, fusion of the cerebral hemispheres, and scalloping of the dentate gyrus. These lesions are associated with abundant astrogliosis and widespread fragmentation of the basal lamina at the cortical surface. During cortical development, neuronal ectopias are associated with severe disorganization of radial glial processes and displacement of Cajal-Retzius cells. Lesions are not seen when Ilk is specifically deleted from embryonic neurons. Interestingly, targeted Ilk deletion has no effect on proliferation or survival of cortical cells or on phosphorylation of two Ilk substrates, Pkb/Akt and Gsk-3β, suggesting that Ilk does not regulate cortical lamination via these enzymes. Instead, Ilk acts in vivo as a major intracellular mediator of integrin-dependent basal lamina formation. This study demonstrates a critical role for Ilk in cortical lamination and suggests that Ilk-associated pathways are involved in the pathogenesis of cobblestone lissencephalies.

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand.

25-Hydroxyvitamin D(3) 1alpha-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), a vitamin D receptor ligand. 25-Hydroxyvitamin D(3) has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1alpha-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D(3) has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D(3) internalisation, in Cyp27b1(-/-) cells explains their higher sensitivity to 25-hydroxyvitamin D(3). 25-Hydroxyvitamin D(3) action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D(3). Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D(3) in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D(3) with 1alpha,25-dihydroxyvitamin D(3) in Cyp27b1(-/-) cells further demonstrates the agonistic action of 25-hydroxyvitamin D(3) and suggests that a synergism between 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) might be physiologically important. In conclusion, 25-hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.