Update on biological actions of 1α,25(OH)2-vitamin D3 (rapid effects) and 24R,25(OH)2-vitamin D3

Molecular and Cellular Endocrinology 197 (2002) 1 /13 www.elsevier.com/locate/mce

Update on biological actions of 1a,25(OH)2-vitamin D3 (rapid effects) and 24R,25(OH)2-vitamin D3 Anthony W. Norman a,*, William H. Okamura b, June E. Bishop a, Helen L. Henry a a

Department of Biochemistry, University of California, Riverside, CA 92521, USA Department of Chemistry, University of California, Riverside, CA 92521, USA

b

Abstract All biologic responses to vitamin D are now known to arise as a consequence of the metabolism of this seco-steroid into its two principal biologically active metabolites 1a,25(OH)2-vitamin D3 (1a,25(OH)2D3) and 24R,25(OH)2-vitamin D3 (24R,25(OH)2D3). 1a,25(OH)2D3 is the dominant metabolite and produces a wide array of biological responses via interacting both with the classical vitamin D nuclear receptor (VDRnuc) that regulates gene transcription in over 30 target organs and with a putative cell membrane receptor (VDRmem1,25) that mediates rapid (within seconds to minutes) biological responses. Ligand occupancy of VDRmem1,25 is linked to signal transduction systems that can mediate the opening of Ca2
and chloride voltage gated channels as well as activation of MAP-kinase. MAP-kinase activation in some cells containing VDRmem1,25
/VDRnuc then results in ‘‘cross-talk’’ from VDRmem1,25 to VDRnuc which modulates transactivation of 1a,25(OH)2D3 responsive gene promoters. The 24R,25(OH)2D3 metabolite has been shown to be an essential hormone for the process of bone fracture healing. The activity of the enzyme responsible for the production of 24R,25(OH)2D3, the renal 25(OH)D-24-hydroxylase, becomes elevated within 4 /11 days after imposition of a tibial fracture, thereby increasing the blood concentrations of 24R,25(OH)2D3 by threefold. The 24R,25(OH)2D3 likely initiates its biological responses via binding to the ligand binding domain of a second cell membrane receptor, the VDRmem24,25, which is stereospecific for 24R,25(OH)2D3 in comparison with 24S,25(OH)2D3 and 1a,25(OH)2D3. This report summarizes the status of several current research frontiers in this arena of the vitamin D endocrine system. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin D; 1a,25(OH)2D3; 24,25(OH)D3; Bone; Fracture healing; Rapid responses

1. Introduction Vitamin D is a precursor molecule to the two biologically active daughter metabolites 1a,25(OH)2vitamin D3 (1a,25(OH)2D3) and 24R,25(OH)2-vitamin D3 (24R,25(OH)2D3) that are both produced in the proximal kidney tubule from the intermediate substrate 25(OH)-vitamin D3 (25(OH)D3). Regulated amounts of both 1a,25(OH)2D3 and 24R,25(OH)2D3 are produced in accordance with various physiological signals operative in the vitamin D endocrine system (see Fig. 1). These two steroid hormones bind to the plasma vitamin D binding protein (DBP) and are delivered systemically to the sites of their respective target organs of the

* Corresponding author. Tel.:
/1-909-787-4777; fax:
/1-909-7874784 E-mail address: [email protected] (A.W. Norman).

vitamin D endocrine system (Henry, 2000; Bouillon et al., 1995). The target organs for the two dihydroxylated vitamin D metabolite hormones are defined by the presence of two types of receptors, namely a nuclear receptor and two membrane receptors (Fig. 1). For the dominant metabolite, there are over 30 known target organs or cell types which possess the classical 50 kDa 1a,25(OH)2D3 nuclear receptor designated as the VDRnuc (Norman and Collins, 2001; Hannah and Norman, 1994). In addition, a second class of receptor for 1a,25(OH)2D3 has been described as being present in the plasma membrane of several cell types (Nemere et al., 1994), which are capable of producing the so-called rapid biological responses (see Table 1). This membrane receptor for 1a,25(OH)2D3 has been designated as VDRmem1,25. With regard to 24R,25(OH)2D3, no convincing evidence has yet been presented describing the presence of a nuclear receptor, but two laboratories

0303-7207/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 2 ) 0 0 2 7 3 - 3

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Fig. 1. Overview of the vitamin D endocrine system. Target organs and cells for 1a,25(OH)2D3 and 24R,25(OH)2D3 by definition contain receptors for one or more of the three classes of receptors that are schematically indicated. 1a,25(OH)2D3 is the ligand for both VDRnuc and VDRmem1,25 while 24R,25(OH)2D3 is the ligand for the VDRmem24,25 receptor.

have reported the existence of a plasma membrane receptor for 24R,25(OH)2D3 in the fracture-healing chick callus (Kato et al., 1998; Seo et al., 1996) and rat resting zone cartilage cells (Pedrozo et al., 1999). The relationships of VDRnuc, VDRmem1,25 and VDRmem24,25 with other classical steroid nuclear receptors has been discussed (Norman, 1998). Vitamin D and all its metabolites, including the steroid hormones 1a,25(OH)2D3 and 24R,25(OH)2D3, in comparison with other steroid hormones, are unusually conformationally flexible (Okamura et al., 1995). This flexibility occurs in three separate regions of the molecule: (a) the side chain with 3608 rotation around the 5 carbon
/carbon single bonds, (b) the broken B-ring with a 3608 rotation around the 6,7 carbon
/carbon bond, and (c) the A-ring where a cyclohexane-like chair l/chair interconversion occurs which changes the orientation of 1a-hydroxyl and 3b-hydroxyl between the

equatorial and axial orientations (Fig. 2). The intrinsic flexibility of these three structural features of vitamin D and its metabolites results in a myriad of shape changes that occur many thousands of times per second. Accordingly, this conformational mobility capability generates a wide array of molecular shapes that, in principle, are available for binding to receptors involved with 1a,25(OH)2D3-mediated biological responses (both genomic and rapid) and 24R,25(OH)2D3 (rapid) and as well for binding to DBP and the substrate binding site of the several vitamin D metabolizing enzymes.

2. 1a,25(OH)2D3 rapid response research frontiers It was originally proposed in 1984 that some actions of 1a,25(OH)2D3 may be mediated at the cell membrane, i.e. by a membrane receptor (Nemere et al.,

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Table 1 Tissue distribution of rapid responses to 1a,25(OH)2D3 Organ/cell/system

Response studied

Reference

Chondrocytes

PKC activation Phospholipase A2 activation PKC effects Subcellular distribution Rapid transport of intestinal Ca2
(transcaltachia) Activation of PKC Activation of MAP-kinase Alter PKC subcellular distribution Activation of highly purified PKC Activation of PKC and MAP-kinase PKC and Ca2
effects ROS 17/2.8 cells Ca2
channel opening Cl  channel opening Phospholipid metabolism Cytosolic Ca2
Aspects of cell differentiation PKC effects Activation of MAP-kinase

Schmitz et al. (1996) Boyan et al. (1998) Bissonnette et al. (1995) Simboli-Campbell et al. (1992) Nemere et al. (1984) Bissonnette et al. (1994) De Boland and Norman (1998) Yada et al. (1989) Slater et al. (1995) Beno et al. (1995) Selle´s and Boland (1991)

Colon Intestine

Keratinocytes Lipid bilayer Liver Muscle Osteoblast

Parathyroid cells Promyelocytic leukemic cells

1984). For the rapid response of transcaltachia (the rapid hormonal stimulation of intestinal calcium transport), a candidate membrane receptor (VDRmem1,25) has been identified and partially purified (Nemere et al., 1994). The 4000X purified protein bound [3H]1,25(OH)2D3 with a Kd :/0.7 /109 M has an estimated molecular weight of 60 kDa (Nemere et al., 1994). Other laboratories have also presented evidence for the existence of a VDRmem1,25 in human leukemic NB4 cells (Berry et al., 1999; Bhatia et al., 1995), intestinal enterocytes (Lieberherr et al., 1989), ROS 24/ 1 cells (Baran et al., 1994), and in chondrocyte matrix vesicles (Pedrozo et al., 1999; Schwartz et al., 1988); in some instances, a partial purification has been effected (Baran et al., 1998). At the present time, VDRmem1,25 1a,25-dihydroxyvitamin D3 must be designated as ‘‘putative’’ since it has not yet been cloned to reveal its biochemical structure. Several reviews of rapid responses to 1a,25(OH)2D3 have appeared (Nemere and Farach-Carson, 1999; Norman, 1997). A wide array of rapid responses stimulated by 1a,25(OH)2D3 have been reported over the past 15 years; a summary is given in Table 1. The most recent additions to the list include demonstration that 1a,25(OH)2D3 can stimulate opening of chloride channels (Zanello and Norman, 1996) and activation of MAP-kinase (Beno et al., 1995; Song et al., 1998). MAPkinase belongs to the family of serine/threonine protein kinases and can be activated by phosphorylation on a tyrosine residue induced by mitogens or cytodifferentiating agents (Pelech and Sanghera, 1992). MAP-kinase integrates multiple intracellular signals transmitted by various second messengers and regulates many cellular functions by phosphorylation of a number of cytoplas-

Caffrey and Farach-Carson (1989) Zanello and Norman (1996) Bourdeau et al. (1990) Sugimoto et al. (1988) Bhatia et al. (1996) Berry et al. (1996) Song et al. (1998)

mic kinases and nuclear transcription factors including the EGF receptor, c-myc and c-jun (Lange-Carter et al., 1993). These rapid actions of 1a,25(OH)2D3 have been postulated to regulate cell biological function and potentially to interact with other membrane-mediated kinase cascades or to cross-talk with the cell nucleus to control genomic responses associated with cell differentiation and proliferation (Berry et al., 1996). One of the hallmarks of 1a,25(OH)2D3 as a steroid hormone is that it is conformationally flexible and is capable of generating a wide array of shapes. This has apparently over evolutionary time allowed generation of more than one type of receptor that can selectively bind different shapes of 1a,25(OH)2D3 as a ligand (see Fig. 4; Norman, 1998). Our laboratories have conducted structure function studies utilizing a wide variety of analogs of 1a,25(OH)2D3, which are locked in a variety of conformational shapes (Norman et al., 1997a). We have presented evidence that the preferred shape for VDRmem1,25, which is linked to rapid responses, is that represented by the 6-s-cis locked planar 1a,25(OH)2lumisterol (JN; see Fig. 3E). Further, as a consequence of developing a molecular model of the ligand binding domain (LBD) of VDRnuc (Norman et al., 1999) and the recent publication of the X-ray crystal structure of VDRnuc-LBD with bound ligand (Rochel et al., 2000), it is now established that the preferred agonist shape of VDRnuc is that represented by a twisted 6-s-trans bowl shape which is close to the 6-s-trans shape of 1a,25(OH)2D3 and analog JB as shown in Fig. 3A and D, respectively (Rochel et al., 2000). Thus we have proposed that VDRnuc and putative VDRmem1,25 utilize different shapes of 1a,25(OH)2D3 as their preferred ligands (Norman et al., 1997a, 2001).

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Fig. 2. Illustration of the conformational flexibility of vitamin D molecules using 1a,25(OH)2D3 as an example. (A) Structure of 1a,25(OH)2D3 with indication of the three structural features of the molecule which confer conformational flexibility. The dynamic rotation of the cholesterol-like side chain of 1a,25(OH)2D3 with 3608 rotations about the 5 single carbon
/carbon bonds are indicated by the curved arrow. (B) 3608 rotational freedom about the 6,7 carbon
/carbon single bond of the seco-B-ring which generates conformations ranging from the more steroid-like (6-s-cis ) conformation to the open and extended (6-s-trans ) conformation of 1a,25(OH)2D3. (C) The rapid (thousands of times per second) chairl/chair interconversions of the A-ring of the seco-steroid which effectively equilibrates the 1a and 3b hydroxyls between the axial and equatorial orientations (Okamura et al., 1995).

Fig. 3F presents an example of rapid responses in rat ROS 17/2.8 osteoblastic cells studied by a whole-cell patch clamping technique; here the conformationally flexible 1a,25(OH)2D3 was discovered to promote the enhancement of outwardly rectifying chloride channels over the course of 1/5 min in a concentration-dependent manner, with a maximum increase of about fourfold between 0.5 and 5 nM of agonist (Zanello and Norman, 1996). Again, it was learned that only 6-s-cis locked analogs like JN were able to stimulate the opening of the chloride channels in a dose-dependent fashion. Thus, the 6-s-trans locked analog JB was found to be ineffective. Finally, analog HL (1b,25(OH)2D3), which has been shown previously to block the rapid responses of 1a,25(OH)2D3 (Norman et al., 1993) also blocked 1a,25(OH)2D3-stimulated opening of chloride channels.

One interesting possibility concerning rapid responses is that occupancy of VDRmem1,25 by a 6-s-cis shaped 1a,25(OH)2D3 results in a process of cross-talk with the nucleus so as to modulate VDRnuc-mediated genomic responses. Both protein kinase C (PKC) and MAPkinase are present in the human promyelocytic leukemia NB4 cells and have been implicated in the process of cell differentiation stimulated by 1a,25(OH)2D3 (Berry et al., 1996; Kraft et al., 1987). In recent studies, we have utilized 6-s-cis locked analogs as a tool to study crosstalk between rapid responses and nuclear response signal transduction pathways. Fig. 3G presents data illustrating that MAP-kinase phosphorylation can be regulated in NB4 cells by 1a,25(OH)2D3 (Song et al., 1998); other studies have clearly implicated MAP-kinase-actuation in altering global gene expression (Roberts et al., 2000). These

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Fig. 3

rapid actions of 1a,25(OH)2D3 could potentially crosstalk with the cell nucleus to control genomic responses associated with cell differentiation and proliferation. Fig. 3G reports the time-dependent effects on MAPkinase activation by the conformationally flexible 1a,25(OH)2D3, the 6-s-cis locked JN and the 6-s-trans locked JB. Only 1a,25(OH)2D3 and JN, but not JB, were

able to activate the MAP-kinase. Fig. 3H presents data supporting the concept of cross-talk from 1a,25(OH)2D3 activation of MAP-kinase to mediate the stimulation of 25(OH)D3-24-hydroxylase gene expression in NB4 cells. Thus, 1a,25(OH)2D3 mediated a significant elevation of the transfected 25(OH)D3-24R-hydroxylase reporter gene. Intriguingly induction of the reporter by

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Fig. 4. Proposed signal transduction mechanisms for 1a,25(OH)2D3-mediated rapid responses. The upper left box lists the rapid responses generated by 1a,25(OH)2D3. 1a,25(OH)2D3 can initiate biologic responses both through VDRnuc and through VDRmem1,25 which generates rapidly the appearance of second messengers, some of which modulate events in the nucleus via cross-talk.

1a,25(OH)2D3 was blocked by application of PD98059, which is a specific blocker of MAP-kinase activation (Pang et al., 1995). These data suggest that the signal transduction event(s) initiated by 1a,25(OH)2D3 for transactivation of the reporter gene were transmitted via activation of the MAP-kinase pathway. Work is

currently in progress to further study this concept in a variety of systems. Fig. 4 presents an integrated model illustrating how different shapes of the conformationally flexible 1a,25(OH)2D3 interact in target cells with both VDRnuc in the nucleus and the plasma membrane localized

Fig. 3. Rapid response research frontiers mediated by 1a,25(OH)2D3 and 6-s-cis locked analogs. (A /E) Structures of important vitamin D related molecules: (A) the steroid hormone 1a,25(OH)2D3; (B) the steroid hormone 24R,25(OH)2D3; (C) analog HL or 1b,25(OH)2D3 which is an antagonist of 1a,25(OH)2D3-mediated rapid responses (Norman et al., 1993); (D) analog JB or 1a,25(OH)2dihydroxyisotachysterol3 which is not an agonist for either VDRnuc or VDRmem1,25; (E) analog JN or 1a,25(OH)2-lumisterol which is a potent ligand for VDRmem1,25-mediated rapid responses (Norman et al., 1997a); (F) comparative effects of analogs of 1a,25(OH)2D3 and other steroids on outward chloride currents in ROS17/2.8 cells. The outward anion current stimulated by 1a,25(OH)2D3 and related structural analogs is measured at 80 mV. The figure also shows the effects obtained with two other steroids, cholesterol and b-estradiol, on the same currents. The concentrations used are: 0.5 nM 1a,25(OH)2D3, 0.5 nM 25(OH)D3, 1 nM HL, 1 nM HL
/0.5 nM 1a,25(OH)2D3, 1 /10 nM JN, 1 /10 nM JB, 50 nM cholesterol and 10 nM b-estradiol. The mean effect of each analog was statistically compared with the effect promoted by 0.5 nM 1a,25(OH)2D3 (* P B/0.05; ** P B/0.01; n/4 /9). The effects of analogs JN and JB were also compared. The data were abstracted from Zanello and Norman (1997). (G) Time-dependent effect of 1a,25(OH)2D3 and 6-s-cis locked analog JN and 6-s-trans locked JB on the phosphorylation of MAP-kinase in NB4 acute promyelocytic leukemia cells. The NB4 cells were treated with the indicated analog at 10 8 M for 1 and 5 min at 37 8C. Vehicle and 1a,25(OH)2D3 at 10 18 M were used as negative and positive controls. The cells were collected after 1 and 5 min. The MAP-kinase phosphorylation was assayed as described in Song et al. (1998). This data was abstracted from Song et al. (1998). (H) Cross-talk from 1a,25(OH)2D3 activation of MAP-kinase to mediate the stimulation 25(OH)D3-24-hydroxylase gene expression induction by 1a,25(OH)2D3 in NB4 cells and its inhibition by the MAP-kinase activation inhibitor, PD-98059. The 25(OH)D3-24hydroxylase promoter was transfected into NB4 cells. After 24 h, the cells were then exposed to 1a,25(OH)2D3, 10 8 M, 9/PD-98059 for 24 h. The total RNA was isolated and the amount of the 25(OH)D3-24-hydroxylase and G3PDH mRNA determined via quantitative reverse transcriptasePCR (RT-PCR) as described in Ozono et al. (1999). The PCR products were analyzed by 2% agarose gel electrophoresis, followed by densitometry. The data were abstracted from Norman et al. (1997b).

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VDRmem1,25 to mediate biological responses. Key aspects of this model include: (a) the existence of two classes of receptors for 1a,25(OH)2D3; (b) the ability of 1a,25(OH)2D3 as a ligand to generate different shapes which satisfy the ligand binding requirements of the two classes of receptors; and (c) the suggestion that rapid responses may have the capability to modulate VDRnuc genomic responses. Also, it is possible that VDRnuc may be involved in the induction process for VDRmem1,25 which would suggest that the presence of rapid responses were dependent on prior interaction of 1a,25(OH)2D3 with its VDRnuc. Or stated differently, 1a,25(OH)2D3-mediated rapid responses could not be observed either in vitamin D-deficient animals (no ligand) or in VDR knockout animals (no receptor). Experiments are currently in progress to study this problem.

3. 24R,25(OH)2D3 research frontiers Unlike the case of 1a,25(OH)2D3, which has a nuclear receptor widely distributed throughout the body which mediates a wide array of biological responses, studies on 24R,25(OH)2D3 have mostly suggested that this secosteroid may produce biological responses in a more limited sphere consisting of cartilage and bone cells, and possibly parathyroid hormone secreting cells. Table 2 summarizes the biological responses that have been reported to be mediated by 24R,25(OH)2D3. The first reports of a biological action that 24R,25(OH)2D3 could generate biological responses, that were unresponsive to 1a,25(OH)2D3, were the inhibition of parathyroid hormone (PTH) secretion in several animal systems (Canterbury et al., 1978; Care et

al., 1977; Chertow et al., 1980) and the regression of hypertrophied parathyroid glands typically present in vitamin D-deficient hypocalcemic chicks (Henry et al., 1977). This was followed by this laboratory’s provocative finding that 24R,25(OH)2D3 was required, in combination with 1a,25(OH)2D3, for normal egg hatchability in both White Leghorn chickens and in Japanese quail (Henry and Norman, 1978). In a follow-up study, it was found that only the naturally occurring 24R,25(OH)2D3 but not the artificial 24S,25(OH)2D3 in combination could support normal egg hatchability in Japanese quail (Norman et al., 1983). However, the bulk of recent reports on 24R,25(OH)2D3 has focused on biological effects in bone or cartilage cells. Healing of rachitic lesions by local administration of 24R,25(OH)2D3 into bone has been reported (Ornoy et al., 1978), while 1a(OH)-vitamin D3 (1a(OH)D3) alone could not prevent rachitic changes in tibial epiphysis of chicks. 24R,25(OH)2D3 given to ovariectomized beagle dogs has been shown to increase bone mass (Nakamura et al., 1992b) and bone strength in rabbits (Nakamura et al., 1992a). The modern era of research on 24R,25(OH)2D3 has focused on the following topics: (a) cell biological studies from the laboratory of Boyan et al. (2001), and (b) the studies of the authors’ laboratories which both describe the preferential in vivo accumulation of [3H]24R,25(OH)2D3 in growth plate cartilage of rats and the discovery of the presence of a membrane receptor for 24R,25(OH)2D3, the VDRmem24,25, present in fracturehealing callus of chicks (Kato et al., 1998; Seo et al., 1996), and (c) the development of the 25(OH)-24hydroxylase knockout mouse (St. Arnaud et al., 1996, 2000). Evidence has also been obtained for a VDRmem24,25 in primary rat cartilage resting zone cells

Table 2 Biological responses attributed to 24R,25(OH)2D3 Organ/cell/system

Response studied

Reference

Bone

Healing of rachitic lesions Increased bone mass in ovariectomized beagle dogs Increased bone strength in rabbits Increased torsional strength in chick tibia Activation of PKC in resting zone chondrocytes

Ornoy et al. (1978) Nakamura et al. (1992b) Nakamura et al. (1992a) Noff et al. (1982) Schwartz et al. (2000)

Inhibits prostaglandin E2 production Increases activity of phospholipase C Combination of 24R,25(OH)2D3
1a,25(OH)2D3 is required for normal egg hatchability in: White Leghorn hens Japanese quail Regression of hypertrophied glands Inhibition of PTH secretion in vivo in dogs

Del Toro et al. (2000) Sylvia et al. (1998)

Chondrocytes-resting zone

Egg hatchability

Parathyroid gland

Pregnancy, human Paget’s disease

7

Inhibition of PTH secretion in vitro Decreased levels of 24R,25(OH)2D3 in maternal sera Decreased levels of 24R,25(OH)2D3 in presence of excessive bone resorption

Henry and Norman (1978) Norman et al. (1983) Henry et al. (1977) Canterbury et al. (1978), Care et al. (1977) Chertow et al. (1980) Hillman et al. (1978) Castro-Errecaborde et al. (1991)

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Fig. 5. 24R,25(OH)2D3 research frontiers: (A) time course of changes in the renal 25(OH)D3-24-hydroxylase activity after chick tibial fracture. Elevation of the renal 25(OH)D3-24-hydroxylase activities were compared between the birds with a tibial fracture and sham groups over (A1) 1 /21 days or (A2) 8 /12 days after imposition of a tibial fracture. Values are mean9/S.E.M. from six individual determinations. *A significance of P B/ 0.001 (t -test) for the difference in the renal 25(OH)D3-24-hydroxylase activity between the birds with a tibial fracture and the shams at 10 days after treatment. The data were abstracted from Seo and Norman (1997)). (B) Fracture healing facilitated by 24R,25(OH)2D3 as evaluated by the determination of bone strength (left panel) and percent bone ash (right panel). Chicks were administered adequate amounts of 25(OH)D3 daily (bar labeled ‘‘continuous’’) as the positive control for the four experimental groups shown in panel (B). In the four experimental groups, 25(OH)D3 was administered for 10 days, and then withdrawn on days 11 /22 when the birds were demonstrably vitamin D metabolite-deficient. On day 22, a tibial fracture was imposed. Then physiological daily doses of 25(OH)D3 (gray bar) 1a,25(OH)2D3 (diagonal bars), or combinations of 1a,25(OH)2D3
/ 24R,25(OH)2D3 (black bar) or 1a,25(OH)2D3
/24S,25(OH)2D3 (cross-hatched bar) were administered on days 22 /40. Bone strength (torsional measurement) and percent bone ash were determined as described in Seo et al. (1997)). 24R,25(OH)2D3 is the naturally occurring hormone in vivo while its epimer 24S,25(OH)2D3 does not occur in vivo. The data were abstracted from Seo et al. (1997)). (C) Evidence for a membrane receptor, VDRmem24,25, for 24R,25(OH)2D3. (C1) Saturation analysis with [3H]24,25(OH)2D3 of the chick callus membrane fractions. The Kd and Bmax were derived from a Scatchard evaluation. (C2) Relative competitive index (RCI) values of vitamin D metabolites with [3H]24,25(OH)2D3 for binding to the chick callus membrane or DBP. The data were derived from a steroid competition assay described in Kato et al. (1998)).

in cell culture (Pedrozo et al., 1999). Also, Boyan et al. have provided the first mechanistic insight into the signal transduction pathways activated in cartilage resting zone cartilage cells by 24R,25(OH)2D3. This includes the activation of PKC via effects on phospholipase A2 (Schwartz et al., 2000), inhibition of prosta-

glandin E production (Del Toro et al., 2000), increasing the activity of phospholipase C (Sylvia et al., 1998) and demonstration of the involvement of arachidonic acid (Boyan et al., 1998). The cytochrome P450 enzyme, the 25(OH)-24-hydryoxylase is responsible both for the in vivo production

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Fig. 5 (Continued )

of 24R,25(OH)2D3 in the kidney and the inactivation of 1a,25(OH)2D3 by its metabolic conversion to 1a,24R,25(OH)3D3. Thus, preparation of mice with a targeted inactivating mutation of the 24-hydroxylase gene (i.e. a knockout of the 25(OH)-24-hydryoxylase gene) represents an innovative approach to study whether the bio-engineered absence of circulating 24R,25(OH)2D3 results in the absence of biological responses that would be predicted to result from the presence of this dihydroxy metabolite. The first report of St. Arnaud et al. (1996) stated that the homozygous 24-hydroxylase knockout mice (///) displayed a phenotype of bone abnormalities characterized by an accumulation of unmineralized matrix at sites of intramembranous ossification, particularly of calvaria and exocortical surfaces of long bones. However, a later report (St. Arnaud et al., 2000) suggested that crossing of the 24-hydroxylase-deficient mice with VDRnuc knockout mice rescued the abnormal bone phenotype,

suggesting that the expression of VDRnuc is necessary for the manifestation of the impaired mineralization phenotype of the 24-hydroxylase (///) mice. One awaits further developments in this system with interest. Some of the current research frontiers related to 24R,25(OH)2D3 studied in the laboratories of the authors are shown in Fig. 5. We tested the hypothesis that 24R,25(OH)2D3 is an essential vitamin D metabolite for the development of normal bone integrity and the healing of fractures. The experimental approach involved a chick model of in vivo fracture healing under circumstances of varying vitamin D metabolite nutritional status. The first consideration was to evaluate whether imposition of a fracture had any discernible consequences on the activity of the kidney enzyme that is responsible for the production of 24R,25(OH)2D3, namely the 25(OH)D3-24R-hydroxylase. As shown in Fig. 5A, there is an approximate threefold elevation in the renal 25(OH)D3-24R-hydroxylase activity that is

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Fig. 6. Proposed mode of action of 24R,25(OH)2D3 for the process of fracture healing. After imposition of a bone fracture, an unknown factor is produced and delivered systemically to the kidney proximal tubule leading to a significant increase in the activity of the 25(OH)D3-24R-hydroxylase. The resultant elevation of the plasma level of 24R,25(OH)2D3 occupies the VDRmem24,25 receptor believed to be present in the fracture-healing callus and thereby initiates signal transduction processes contributing to the process of fracture healing.

first discernible 3/7 days after imposition of the fracture (Fig. 5A1) and which becomes maximal over the time period of 8/12 days after the fracture (Fig. 5A2). In data not shown here, but discussed in reference (Seo and Norman, 1997), the presence of the fracture did not alter the activity of the 25(OH)D3-1a-hydroxylase. Consistent with these results was the observation that the plasma levels of 24R,25(OH)2D3 were 3.6-fold higher in the serum of birds 10 days after imposition of a tibial fracture as compared with sham controls; over the same time period, there was no change in the serum levels of 1a,25(OH)2D3 (Seo and Norman, 1997). Fracture-healing results facilitated by 24R,25(OH)2D3, as evaluated by the determination of bone strength (left panel) and percent bone ash (right panel) are shown in Fig. 5B. The naturally occurring 24R,25(OH)2D3 and its synthetic epimer 24S,25(OH)2D3 were tested alone or in combination with 1a,25(OH)2D3, on normal bone development and other related variables of the Ca2
homeostasis system (serum Ca2
, 25(OH)D3, 24,25(OH)2D3, 1a,25(OH)2D3 levels) in chicks. Mechanical testing of torsional strength was carried out on the femur (Seo et al., 1997). In

control studies, 24R,25(OH)2D3 (80 nmol/kg diet) alone was found to be sufficient for normal bone growth and integrity similar to that achieved by the vitamin D3replete controls. Accordingly, in the experimental results shown in Fig. 5B, chicks were fed a 25(OH)D3-replete diet (75 nmol/kg diet) for 8 days after hatching and 25(OH)D3 was then withdrawn to minimize any residual circulating metabolites prior to the imposition of standardized tibial fractures 14 days later. Vitamin D metabolites were then administered for 2 weeks to determine their effects on the mechanical properties of healed tibiae. 24S,25(OH)2D3 combined with 1a,25(OH)2D3 or 1a,25(OH)2D3 alone resulted in poor bone strength (48 and 39%, respectively) and reduced bone ash (78 and 66%, respectively) compared with the control group (100%). In contrast, the fractured tibiae of the birds fed 24R,25(OH)2D3 in combination with 1a,25(OH)2D3 showed a bone strength 92% and bone ash 93% of the control group. These results suggest that when 24R,25(OH)2D3 is present at normal physiological concentrations, it is an essential vitamin D3 metabolite for both normal bone integrity and healing of fractures in chicks.

A.W. Norman et al. / Molecular and Cellular Endocrinology 197 (2002) 1 /13

In light of the ability of the fracture-healing callus to discriminate between 24R,25(OH)2D3 and 24S,25(OH)2D3, a search was initiated in fracturehealing callus tissue for the presence of a specific 24R,25(OH)2D3 receptor. No evidence was obtained for a classical nuclear/cytosol receptor for 24R,25(OH)2D3 in the callus (Kato et al., 1998). Evidence for a specific saturable receptor binding protein for 24R,25(OH)2D3 was found in the callus membrane fraction which displayed a KD /18.39/1.9 nmol/l (Fig. 5C1). The binding of the [3H]24R,25(OH)2D3 to the callus membrane receptor binding protein was specific in that it could only be competed by nonradioactive 24R,25(OH)2D3 but not by 24S,25(OH)2D3, 1a,25(OH)2D3 or 25(OH)D3 (Fig. 5D2). Also shown in Fig. 5C2 is the clear difference in ligand specificities, displayed by VDRmem24,25 and the plasma DBP, particularly between 24R,25(OH)2D3 and 24S,25(OH)2D (Kato et al., 1998). Based on these observations, it is possible to envision the existence of an endocrine system relating a bone fracture to the kidney 25(OH)D3-24R-hydroxylase, resulting in the subsequent elevation of the serum 24R,25(OH)2D3 levels. The increased availability of this steroid hormone allows occupancy of the proposed VDRmem24,25 receptor which then initiates, in collaboration, with 1a,25(OH)2D3, of appropriate signal transduction signals that orchestrate the competent healing of the fracture (see Fig. 6). Preliminary evidence supporting the existence of a serum-borne chemical messenger from the fracture site to the kidney has been obtained. Studies are also currently in progress to identify the nature of the cellular interactions and the details of the involved signal transduction pathway related to the biological actions of 24R,25(OH)2D3.

4. Summary This paper has focused on current challenging research areas in the vitamin D endocrine system with particular emphasis on the aspects of how 1a,25(OH)2D3 mediates rapid biological responses and how 24R,25(OH)2D3 produces unique biological responses. While both these research topics are considered by some to be controversial, it is only through the conduct of further critical experiments that the necessary data to place these two concepts on a firm scientific ground will be obtained.

Acknowledgements Parts of this work were supported by a grant from the USPHS DK09012-35 (AWN) and DK-16595 (WHO).

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