Infantile hypercalcemia type 1 (HCINF1): a rare disease resulting in nephrolithiasis and nephrocalcinosis caused by mutations in the vitamin D catabolic enzyme, CYP24A1.

Infantile hypercalcemia type 1 (HCINF1), formerly known as Lightwood syndrome, is a subtype of hypercalcemia caused by loss-of-function biallelic mutations in the vitamin D catabolic enzyme, CYP24A1, which 24-hydroxylates the hormone 1,25-(OH)2D3. This short review focuses on the main features of the HCINF1 disease; emerging knowledge of the structure and function of the cytochrome P450, CYP24A1 and the location of inactivating mutations; the development of a rapid LC-MS/MS-based laboratory test for defective 24-hydroxylation; and future implications for bioanalytical assay and treatment of all types of vitamin D-related hypercalcemic conditions.

Integrin-linked kinase has a critical role in ErbB2 mammary tumor progression: implications for human breast cancer.

Elevated expression of the integrin-linked kinase (ILK) has been observed in a variety of cancers and has been further correlated with poor clinical outcome. Here, we show that mammary epithelial disruption of ILK results in a profound block in mammary tumor induction. Consistent with these observations, inhibition of ILK function in ErbB2-expressing cells with small molecule inhibitor or RNA interference resulted in profound block in their in vitro invasive properties due to the induction of apoptotic cell death. The rare ILK-deficient tumors that eventually arose overcame this block in tumor induction by an upregulation of ErB3 phosphorylation. These observations provide direct evidence that ILK has a critical role in the initiation phase of ErbB2 tumor induction.

1α,24(S)(OH)2D2 normalizes bone morphology and serum parathyroid hormone without hypercalcemia in 25-hydroxyvitamin D-1-hydroxylase (CYP27B1)-deficient mice, an animal model of vitamin D deficiency with secondary hyperparathyroidism.

BACKGROUND: Vitamin D compounds are effective in managing elevated PTH levels in secondary hyperparathyroidism (SHPT) of renal failure. However, undesired increases in serum calcium and phosphorus associated with compounds such as calcitriol [1,25(OH)2D3] has prompted a search for compounds with improved safety profiles. 1alpha,24(S)(OH)2D2 (1,24(OH)2D2) is a vitamin D2 metabolite with low calcium-mo bilizing activity in vivo. We studied the efficacy of 1,24(OH)2D2 in mice lacking the CYP27B1 enzyme [25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase)], a novel vitamin D deficiency model with SHPT. MATERIALS AND METHODS: 1alpha-OHase-deficient (-/-) mice and normal (+/-) heterozygous littermates re ceived 1,24(OH)2D2 (100, 300, 1000, and 3000 pg/g/day) or 1,25(OH)2D3 (30, 300, and 500 pg/g/day) for 5 weeks via daily sc injection. Control groups received vehicle. RESULTS: Vehicle-treated 1alpha-OHase-deficient mice were hypocalcemic and had greatly elevated serum PTH. 1,24(OH)2D2 at doses above 300 pg/g/day normalized serum calcium, serum PTH, bone growth plate morphology, and other bone parameters. No hy percalcemia was observed at any dose of 1,24(OH)2D2 in normal or 1alpha-OHase-deficient animals. In contrast, 1,25(OH)2D3 at only 30 pg/g/day normalized calcemia, serum PTH, and bone parameters, but at higher doses completely suppressed PTH and caused hypercalcemia in both 1alpha-OHase-deficient and normal mice. Treatment with 500 pg/g/day of 1,25(OH)2D3 also induced osteomalacia in normal animals. CONCLUSION: 1,25(OH)2D3 was maximally active at 10-fold lower doses than 1,24(OH)2D2, but induced hypercalcemia and osteomalacia at high doses. 1,24(OH)2D2 normalized serum calcium, serum PTH, and bone histomorphometry without hypercalcemia in 1alpha-OHase-deficient mice with SHPT.

Integrin-linked kinase regulates cell proliferation and tumour growth in murine colitis-associated carcinogenesis.

BACKGROUND: Integrins are transmembrane cell surface receptors that mediate cell-cell and cell-matrix contacts. Integrin-linked kinase (ILK) is the binding partner of beta1 and beta3 integrins, and has been ascribed essential roles in development, angiogenesis and tumourigenesis. However, in vivo evidence for the latter is currently lacking. AIM: The hypothesis that epithelial cell-specific deletion of ILK would impact on murine tumourigenesis was tested using a colitis-associated cancer model. METHODS: To create intestinal epithelial cell ILK knockout animals, Fabp/Cre mice (Cre recombinase expressed under the control of a modified Fabp promoter) were used, and they were mated with mice carrying a loxP-flanked (floxed) ILK gene (ILK(flox/flox)). RESULTS: ILK intestinal knockout mice exhibited a reduction in the size of the caecum, and reduced crypt height in the colon. Immunohistochemical analysis confirmed that there was diminished ILK expression, and bromodeoxyuridine (BrdU) staining was significantly reduced in the knockout animals as compared with the wild-type animals in both the caecum and colon (p<0.001 for both). Following azoxymethane and dextran sodium sulfate (DSS) treatment, fewer total tumours were observed in the ILK knockout animals, which were mosaic with respect to ILK expression. Cyclin D1, Snail, fibronectin and matrix metalloproteinase 9 (MMP9) were all reduced, and active caspase 3 increased, in tumours from ILK knockout mice, as compared with wild-type mice, on immunohistochemical analysis. Using small interfering RNA (siRNA) to knock down ILK in colonic cancer cell lines, it was confirmed that it is capable of regulating cyclin D1, Snail, MMP9 and fibronectin transcription. CONCLUSIONS: From these findings, it is concluded that ILK plays an important role in intestinal epithelial cell proliferation, and that it influences the development of colitis-associated cancer, through modulation of cyclin D1, the extracellular matrix and MMP9.

Differential mechanisms of transcriptional regulation of the mouse osteocalcin gene by Jun family members.

The osteocalcin gene encodes an osteoblast-specific protein that is induced with the onset of mineralization at late stages of differentiation. Several transcriptional regulators have been characterized that control the transcription of osteocalcin, including activator protein 1 (AP-1) family members such as the Fra2/JunD heterodimer. We have previously shown that the c-Jun homodimer activates transcription from the murine osteocalcin proximal promoter and that this response is potentiated by the alpha chain of the nascent polypeptide-associated complex (alphaNAC) transcriptional coactivator. We now further explore the mechanisms involved and show that c-Jun binds two cryptic AP-1 sites within the proximal promoter of osteocalcin and that this binding is strictly alphaNAC-dependent. Chromatin immunoprecipitation (ChIP) confirmed that c-Jun occupies its binding sites within the osteocalcin 5'-flanking region in living osteoblasts. Interestingly, the ChIP assay revealed that both JunB and JunD also bind the osteocalcin promoter. JunD, but not JunB, stimulated osteocalcin gene transcription in transient transfection assays, but this effect was not potentiated by alphaNAC. Thus, the c-Jun and JunD family members utilize distinct mechanisms that implicate differential interaction with transcriptional coactivators to regulate osteocalcin expression.

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-1alpha-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.

HES6 acts as a transcriptional repressor in myoblasts and can induce the myogenic differentiation program.

HES6 is a novel member of the family of basic helix-loop-helix mammalian homologues of Drosophila Hairy and Enhancer of split. We have analyzed the biochemical and functional roles of HES6 in myoblasts. HES6 interacted with the corepressor transducin-like Enhancer of split 1 in yeast and mammalian cells through its WRPW COOH-terminal motif. HES6 repressed transcription from an N box-containing template and also when tethered to DNA through the GAL4 DNA binding domain. On N box-containing promoters, HES6 cooperated with HES1 to achieve maximal repression. An HES6-VP16 activation domain fusion protein activated the N box-containing reporter, confirming that HES6 bound the N box in muscle cells. The expression of HES6 was induced when myoblasts fused to become differentiated myotubes. Constitutive expression of HES6 in myoblasts inhibited expression of MyoR, a repressor of myogenesis, and induced differentiation, as evidenced by fusion into myotubes and expression of the muscle marker myosin heavy chain. Reciprocally, blocking endogenous HES6 function by using a WRPW-deleted dominant negative HES6 mutant led to increased expression of MyoR and completely blocked the muscle development program. Our results show that HES6 is an important regulator of myogenesis and suggest that MyoR is a target for HES6-dependent transcriptional repression.

Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets.

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.

Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

Heart, brain, and body wall defects in mice lacking calreticulin.

Calreticulin is a ubiquitously expressed protein, which has been implicated in a large number of cellular functions, including calcium storage and signaling, protein folding, and cell attachment. To examine the role of calreticulin during in vivo development, mice deficient in calreticulin were generated by targeted inactivation of the calreticulin gene. Calreticulin-deficient mutants die in utero, mostly in late gestation. Half of these embryos had decreased cardiac cell mass, associated with increased apoptosis of cardiac myocytes. In vitro differentiation cultures of calreticulin-deficient embryonic stem cells resulted in fewer embryoid bodies with contractile activity than cultures derived from calreticulin +/- stem cells (P < 0.001). Sixteen percent of the mutants exhibited exencephaly secondary to a defect in neural tube closure. Embryos surviving until Embryonic Day 16.5 had omphalocele. Lack of calreticulin did not influence survival of embryonic fibroblasts under various endoplasmic reticulum stress conditions. However, calreticulin did influence cell migration in a calcium- and substrate-dependent manner. We conclude that calreticulin is not essential during the early stages of embryonic development, but is important for the development of heart and brain and for ventral body wall closure. The observed abnormalities are compatible with a role of calreticulin in the modulation of cellular calcium signaling.

The 3-epi- and 24-oxo-derivatives of 1alpha,25 dihydroxyvitamin D(3) stimulate transcription through the vitamin D receptor.

Vitamin D is enzymatically modified to more than 35 metabolites. While many of these are thought to represent degradation products, some have been shown to exhibit biological activity. We tested whether 3-epi-1alpha,25-dihydroxyvitamin D(3) (3-epi-1alpha, 25(OH)(2)D(3)), 1alpha,25-dihydroxy-24-oxo-vitamin D(3) (1alpha, 25(OH)(2)-24-oxo-D(3)), and 1alpha,25(OH)(2)D(3)-26,23-lactone can stimulate transcription of vitamin D responsive genes. MC3T3-E1 cells transfected with a 25-hydroxyvitamin D 24-hydroxylase (CYP24) promoter construct displayed a 6 fold response when treated with either 1alpha,25(OH)(2)D(3) or 3-epi-1alpha,25(OH)(2)D(3). Caco-2 cells were transfected with the wild type CYP24 promoter construct, or a Vitamin D Response Element (VDRE)-mutated form. Cells acquiring the wild type reporter responded to 1alpha,25(OH)(2)D(3) and 3-epi-1alpha,25(OH)(2)D(3) but not cells which acquired the mutated reporter. Additionally, VDR-negative COS-7 cells transfected with the wild type promoter responded (approximately 13 fold) to 1alpha, 25(OH)(2)D(3) and 3-epi-1alpha,25(OH)(2)D(3), only when co-transfected with the VDR. These results were confirmed using shorter incubation times and serum-free conditions. This strongly suggested that 3-epi-1alpha,25(OH)(2)D(3) mediates its effects through the VDR and its cognate binding site. Similar results were obtained with 1alpha,25(OH)(2)-24-oxo-D(3) using VDR-negative P19 cells. We could never detect activity from 1alpha,25(OH)(2)D(3)-26, 23-lactone on vitamin D-responsive target promoters. Our results firmly conclude that both 3-epi-1alpha,25(OH)(2)D(3) and the 1alpha, 25(OH)(2)-24-oxo-D(3) elicit their biological effects by acting through the VDR/VDRE.

Novel findings about 24,25-dihydroxyvitamin D: an active metabolite?

The physiological role of 24,25-dihydroxyvitamin D remains controversial. Recent results suggest that 24,25-dihydroxyvitamin D is essential for fracture healing, and binding sites for 24,25-dihydroxyvitamin D have been identified in fracture callus tissue. Mice deficient in the 25-hydroxyvitamin D-24-hydroxylase enzyme provide novel genetic tools in which to study the role of 24,25-dihydroxyvitamin D in bone development and fracture repair.

Targeted inactivation of vitamin D hydroxylases in mice.

Vitamin D undergoes a first hydroxylation in the liver to generate 25-hydroxyvitamin D, then this metabolite is further hydroxylated in the kidney to yield either 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], or 24R,25-dihydroxyvitamin D[24,25(OH)2D]. The production of 1alpha,25(OH)2D is catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase), while the synthesis of 24,25(OH)2D is catalyzed by the enzyme 25-hydroxyvitamin D-24-hydroxylase (24-OHase). To determine the role of each of these enzymes in vivo and their putative role during development, we have inactivated each gene by homologous recombination in embryonic stem cells. The targeting vector for the 1alpha-OHase gene was constructed to allow tissue-specific gene inactivation in order to study the hypothesized paracrine/autocrine roles of the 1alpha-OHase enzyme in particular target tissues such as skin, brain, or macrophages. The targeting vector for the 24-OHase gene utilized standard methodology, and analysis of the phenotype of 24-OHase-deficient mice confirmed the role of the 24-OHase enzyme in the catabolism of 1alpha,25(OH)2D. The phenotype of the second generation 24-OHase-null mice also suggests a key role for 24,25(OH)2D in intramembranous bone formation during development.

Dual functions for transcriptional regulators: myth or reality?

Several transcriptional regulatory molecules have been described that appear to possess dual function in separate cellular compartments. It remains unclear whether the proteins really exert dual functions, or which of the transcriptional regulatory role or the cytoplasm-associated function is the physiologically relevant action of the protein. This review will briefly describe the cases at hand and attempt to sort the true bifunctional proteins from the aritfactual trespassers. J. Cell. Biochem. Suppls. 32/33:32-40, 1999.

Molecular cloning of (25-OH D)-1 alpha-hydroxylase: an approach to the understanding of vitamin D pseudo-deficiency.

Pseudovitamin D-deficiency rickets (PDDR) is the first identified inborn error of vitamin D metabolism. Its clinical course is similar to that of nutritional rickets due to simple vitamin D deficiency. The treatment of choice is replacement therapy with calcitriol [1,25(OH)2D3]. PDDR is inherited as a simple autosomal recessive trait. The PDDR locus has been mapped to chromosome 12q13-q14. The molecular defect underlying the 25-hydroxyvitamin D-1 alpha-hydroxylase enzyme dysfunction has remained elusive due to the lack of sequence information for the gene encoding the cytochrome P450 moiety of the enzyme. We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase sequence to identify and clone the 1 alpha-OHase cDNA. The candidate gene was transiently expressed in P19 embryonal carcinoma cells. Only those cells that were transfected with the candidate cDNA in the sense orientation were able to produce a compound that co-eluted with the 1 alpha, 25 vitamin D3 standard. Mass spectrometry analysis confirmed the identity of the produced metabolite. A human genomic clone was isolated from a chromosome 12 cosmid library and subsequently mapped to human chromosome 12q13.1-q13.3. To address the putative biological function of 24,25-dihydroxyvitamin I) 24,25(OH)2D, we also engineered a null mutation in the 24-OHase gene in embryonic stem cells (ES). Animals heterozygous for the engineered mutation are normal and fertile. One half of the homozygous animals die before weaning. Breeding of surviving females gives an F2 generation in which bone development is abnormal at sites of intramembranous ossification. Growthplate maturation and endochondral ossification appeared to proceed normally. The results show that a complete absence of vitamin D metabolites hydroxylated in position 24 during embryogenesis leads to abnormal bone structure and suggests a key role for 24,25(OH)2D in the developmental regulation of intramembranous ossification.

Transcriptional regulation during mesenchymal cell differentiation: the role of coactivators.

We have begun to understand the molecular mechanisms involved in the differentiation of pluripotent mesenchymal stem cells through the identification and characterization of the sequence-specific DNA-binding transcriptional activators that control differentiation along specific lineages. However, recent progress in the study of the mechanisms of gene transcription has identified an additional class of proteins essential for activated gene transcription, namely, the coactivators (sometimes also referred to as adaptors, mediators, or integrator molecules). This review focuses on the identified coactivators that could play a regulatory role in the differentiation of mesenchymal precursors along the myogenic and osteoblastic programs. Interestingly, one such coactivator specifically expressed in bone cells during development, alpha NAC (Nascent-polypeptide-associated complex And Coactivator alpha), is converted into a DNA-binding activator by differential splicing in differentiated myotubes. This suggests that NAC isoforms may be involved in multiple steps along the lineage-making decisions facing pluripotent mesenchymal precursors.

Transcriptional coactivators potentiating AP-1 function in bone.

The AP-1 proteins are formed by the heterodimerization of Fos family members and Jun family members through a structural motif called the leucine zipper. The heterodimer can then bind DNA at a consensus site termed the AP-1 site and act as a transcription factor to modulate the expression of AP-1-responsive genes. All the Jun family members can also homodimerize to exert the same function. Genetic studies including gain-of-function and loss-of-function mutations have shown that AP-1 components, particularly the c-Fos protein, are essential for proper bone development. Both Fos and Jun family members interact with coactivator molecules to activate transcription. To date, the coactivator proteins CBP (CREB-binding protein), JAB1 (Jun-activation domain-binding protein 1), and alpha-NAC (Nascent polypeptide associated complex And Coactivator alpha) have been shown to potentiate the AP-1 transcriptional activating function. We have shown that all three proteins are expressed in bone during mouse development. These findings raise the intriguing possibility that multiple coactivators may be involved in mediating AP-1-dependent transcription and increase the specificity of target gene activation by AP-1 proteins in differentiating bone cells.

Bone-specific expression of the alpha chain of the nascent polypeptide-associated complex, a coactivator potentiating c-Jun-mediated transcription.

The alpha chain of the nascent polypeptide-associated complex (alpha-NAC) coactivator was shown to potentiate the activity of the homodimeric c-Jun activator, while transcription mediated by the c-Fos/c-Jun heterodimer was unaffected. The use of deletion mutants in pull-down assays revealed that alpha-NAC interacted with amino acids 1 to 89 of the c-Jun protein and that the coactivator could interact with both the unphosphorylated and the serine 73-phosphorylated form of c-Jun. N-terminal-deleted c-Jun protein failed to interact with alpha-NAC in mammalian two-hybrid assays, while mutant c-Jun proteins lacking the leucine zipper or the basic domain retained interaction with alpha-NAC in vivo. Kinetics studies with purified c-Jun homodimer and recombinant alpha-NAC proteins allowed determination of the mechanism of coactivation by alpha-NAC: the coactivator stabilized the AP-1 complex formed by the c-Jun homodimer on its DNA recognition sequence through an eightfold reduction in the dissociation constant (kd) of the complex. This effect of alpha-NAC was specific, because alpha-NAC could not stabilize the interactions of JunB or Sp1 with their cognate binding sites. Interestingly, the expression of alpha-NAC was first detected at 14.5 to 15 days postconception, concomitantly with the onset of ossification during embryogenesis. The alpha-NAC protein was specifically expressed in differentiated osteoblasts at the centers of ossification. Thus, the alpha-NAC gene product exhibits the properties of a developmentally regulated, bone-specific transcriptional coactivator.