Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia

Vitamin D, the major steroid hormone that controls mineral ion homeostasis, exerts its actions through the vitamin D receptor (VDR). The VDR is expressed in many tissues, including several tissues not thought to play a role in mineral metabolism. Studies in kindreds with VDR mutations (vitamin D-dependent rickets type II, VDDR II) have demonstrated hypocalcemia, hyperparathyroidism, rickets, and osteomalacia. Alopecia, which is not a feature of vitamin D deficiency, is seen in some kindreds. We have generated a mouse model of VDDR II by targeted ablation of the second zinc finger of the VDR DNA-binding domain. Despite known expression of the VDR in fetal life, homozygous mice are phenotypically normal at birth and demonstrate normal survival at least until 6 months. They become hypocalcemic at 21 days of age, at which time their parathyroid hormone (PTH) levels begin to rise. Hyperparathyroidism is accompanied by an increase in the size of the parathyroid gland as well as an increase in PTH mRNA levels. Rickets and osteomalacia are seen by day 35; however, as early as day 15, there is an expansion in the zone of hypertrophic chondrocytes in the growth plate. In contrast to animals made vitamin D deficient by dietary means, and like some patients with VDDR II, these mice develop progressive alopecia from the age of 4 weeks.

Oligomerization of activated d- and l-guanosine mononucleotides on templates containing d- and l-deoxycytidylate residues

The oligomerization of activated d- and l- and racemic guanosine-5′-phosphoro-2-methylimidazole on short templates containing d- and l-deoxycytidylate has been studied. Results obtained with d-oligo(dC)s as templates are similar to those previously reported for experiments with a poly(C) template. When one l-dC or two consecutive l-dCs are introduced into a d-template, regiospecific synthesis of 3′-5′ oligo(G)s proceeds to the end of the template, but three consecutive l-dCs block synthesis. Alternating d-,l-oligomers do not facilitate oligomerization of the d-, l-, and racemic 2-guanosine-5′-phosphoro-2-methylimidazole. We suggest that once a “predominately d-metabolism” existed, occasional l-residues in a template would not have led to the termination of self-replication.

Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.

Sequential Actions of Phospholipase D and Phosphatidic Acid Phosphohydrolase 2b Generate Diglyceride in Mammalian Cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1–enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.

Increased CNS levels of apolipoprotein D in schizophrenic and bipolar subjects: Implications for the pathophysiology of psychiatric disorders

Chronic administration of the atypical antipsychotic drug, clozapine, to rodents has been shown to increase the concentration of apolipoprotein D (apoD) in several area of the brain, suggesting that apoD could be involved in the therapeutic effects of antipsychotic drugs and/or the pathology of psychotic illnesses. Here, we measured a significant decrease in the concentration of apoD in serum samples from schizophrenic patients. In contrast, apoD levels were significantly increased (92–287%) in dorsolateral prefrontal cortex (Brodmann's area 9) of schizophrenic and bipolar subjects. Elevated levels of apoD expression were also observed in the caudate of schizophrenic and bipolar subjects (68–89%). No differences in apoD immunoreactivity were detected in occipital cortex (Brodmann's area 18) in either group, or in the hippocampus, substantia nigra, or cerebellum of the schizophrenic group. The low serum concentrations of apoD observed in these patients supports recent hypotheses involving systemic insufficiencies in lipid metabolism/signaling in schizophrenia. Elevation of apoD expression selectively within central nervous system regions implicated in the pathology of these neuropsychiatric disorders suggests a focal compensatory response that neuroleptic drug regimens may augment.

Subcellular Distribution and Tissue Expression of Phospholipase D��, D��, and D�� in Arabidopsis1

Three phospholipase Ds (PLDs; EC 3.1.4.4) have been cloned from Arabidopsis, and they exhibit two distinct types of activities: polyphosphoinositide-requiring PLD�� and PLD��, and polyphosphoinositide-independent PLD��. In subcellular fractions of Arabidopsis leaves, PLD�� and PLD�� were both present in the plasma membrane, intracellular membranes, mitochondria, and clathrin-coated vesicles, but their relative levels differed in these fractions. In addition, PLD�� was detected in the nuclear fraction. In contrast, PLD�� was not detectable in any of the subcellular fractions. PLD�� activity was higher in the metabolically more active organs such as flowers, siliques, and roots than in dry seeds and mature leaves, whereas the polyphosphoinositide-dependent PLD activity was greater in older, senescing leaves than in other organs. PLD�� mRNA accumulated at a lower level than the PLD�� and PLD�� transcripts in most organs, and the expression pattern of the PLD�� mRNA also differed from that of PLD�� and PLD�� in different organs. Collectively, these data demonstrated that PLD��, PLD��, and PLD�� have different patterns of subcellular distribution and tissue expression in Arabidopsis. The present study also provides evidence for the presence of an additional PLD that is structurally more closely related to PLD�� than to the other two PLDs.

Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity.

Alterations of various components of the cell cycle regulatory machinery that controls the progression of cells from a quiescent to a growing state contribute to the development of many human cancers. Such alterations include the deregulated expression of G1 cyclins, the loss of function of activities such as those of protein p16INK4a that control G1 cyclin-dependent kinase activity, and the loss of function of the retinoblastoma protein (RB), which is normally regulated by the G1 cyclin-dependent kinases. Various studies have revealed an inverse relationship in the expression of p16INK4a protein and the presence of functional RB in many cell lines. In this study we show that p16INK4a is expressed in cervical cancer cell lines in which the RB gene, Rb, is not functional, either as a consequence of Rb mutation or expression of the human papillomavirus E7 protein. We also demonstrate that p16INK4a levels are increased in primary cells in which RB has been inactivated by DNA tumor virus proteins. Given the role of RB in controlling E2F transcription factor activity, we investigated the role of E2F in controlling p16INK4a expression. We found that E2F1 overexpression leads to an inhibition of cyclin D1-dependent kinase activity and induces the expression of a p16-related transcript. We conclude that the accumulation of G1 cyclin-dependent kinase activity during normal G1 progression leads to E2F accumulation through the inactivation of RB, and that this then leads to the induction of cyclin kinase inhibitor activity and a shutdown of G1 kinase activity.

A possible role of vitamin D receptors in regulating vitamin D activation in the kidney.

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases. Previously, we reported that renal proximal convoluted tubules, the major site of 1 alpha, 25-dihydroxyvitamin D3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1 alpha, 25-dihydroxyvitamin D3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities. In rat models of enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3, expression of vitamin D receptors and 25-hydroxyvitamin D3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts. In vitamin D-deficient rats with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity, expression of vitamin D receptor mRNA in renal proximal convoluted tubules was also down-regulated, indicating that the down-regulation of vitamin D receptor mRNA is not the result of the enhanced production of 1 alpha, 25-dihydroxyvitamin D3. In Japanese quail models with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by sex steroids, expression of vitamin D receptor mRNA was also down-regulated in the kidney but not in the duodenum. These results suggest that the down-regulation of vitamin D receptors plays a critical role in production of 1 alpha, 25-dihydroxyvitamin D3 in renal proximal convoluted tubules.

Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

Mg-chelatase of tobacco: The role of the subunit CHL D in the chelation step of protoporphyrin IX

The Mg-chelation is found to be a prerequisite to direct protoporphyrin IX into the chlorophyll (Chl)-synthesizing branch of the tetrapyrrol pathway. The ATP-dependent insertion of magnesium into protoporphyrin IX is catalyzed by the enzyme Mg-chelatase, which consists of three protein subunits (CHL D, CHL I, and CHL H). We have chosen the Mg-chelatase from tobacco to obtain more information about the mode of molecular action of this complex enzyme by elucidating the interactions in vitro and in vivo between the central subunit CHL D and subunits CHL I and CHL H. We dissected CHL D in defined peptide fragments and assayed for the essential part of CHL D for protein–protein interaction and enzyme activity. Surprisingly, only a small part of CHL D, i.e., 110 aa, was required for interaction with the partner subunits and maintenance of the enzyme activity. In addition, it could be demonstrated that CHL D is capable of forming homodimers. Moreover, it interacted with both CHL I and CHL H. Our data led to the outline of a two-step model based on the cooperation of the subunits for the chelation process.

Cloning and mitochondrial localization of full-length D-AKAP2, a protein kinase A anchoring protein

Differential compartmentalization of signaling molecules in cells and tissues is being recognized as an important mechanism for regulating the specificity of signal transduction pathways. A kinase anchoring proteins (AKAPs) direct the subcellular localization of protein kinase A (PKA) by binding to its regulatory (R) subunits. Dual specific AKAPs (D-AKAPs) interact with both RI and RII. A 372-residue fragment of mouse D-AKAP2 with a 40-residue C-terminal PKA binding region and a putative regulator of G protein signaling (RGS) domain was previously identified by means of a yeast two-hybrid screen. Here, we report the cloning of full-length human D-AKAP2 (662 residues) with an additional putative RGS domain, and the corresponding mouse protein less the first two exons (617 residues). Expression of D-AKAP2 was characterized by using mouse tissue extracts. Full-length D-AKAP2 from various tissues shows different molecular weights, possibly because of alternative splicing or posttranslational modifications. The cloned human gene product has a molecular weight similar to one of the prominent mouse proteins. In vivo association of D-AKAP2 with PKA in mouse brain was demonstrated by using cAMP agarose pull-down assay. Subcellular localization for endogenous mouse, rat, and human D-AKAP2 was determined by immunocytochemistry, immunohistochemistry, and tissue fractionation. D-AKAP2 from all three species is highly enriched in mitochondria. The mitochondrial localization and the presence of RGS domains in D-AKAP2 may have important implications for its function in PKA and G protein signal transduction.

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.