Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 4871-4875, December 1975 Biochemistry

Cytoplasmic and nuclear binding components for la,25-dihydroxyvitamin D3 in chick parathyroid (/receptors/parathyroid ) PETER F. BRUMBAUGH, MARK R. HUGHES, AND MARK R. HAUSSLER Department of Biochemistry, College of Medicine, University of Arizona, Tucson, Ariz. 85724 Communicated by John H. Northrop, September 29, 1975

ABSTRACT Specific binding of la,25-dihydroxyvitamin role of 1a,25-(OH)2D3 in the homeostatic regulation of cal- D3 [la,25(OH)2D3J to macromolecular components in the cy- cium and phosphate can be elucidated. toplasm and nucleus is demonstrated in parathyroid glands Henry and Norman (14) have recently reported that of vitamin-D-deficient chicks. The interaction of la,25- (OH)WD3 with the cytoplasmic binding component is of high la,25-(OH)2[3H]Da is localized in chick parathyroid glands affinity (R4 = 3.2 X 10-10 M) and high specificity [la,25- following administration of the sterol, in vivo. In the present (OH)2D3 > 25-hydroxyvitamin D3 > la-hydroxyvitamin D3 > experiments, specific binding components for 1a,25- vitamin D3 in competing with radioactive la,25(0H)2D31. (OH)2D3 have been isolated from chick parathyroid glands Both cytoplasmic and nuclear hormone-macromolecular and characterized in vitro. Previous reports (15, 16) have complexes sediment at 3.1 S in 0.3 M KCI-sucrose gradients, demonstrated that 1la,25-(OH)2D3 interacts with the intes- and agarose gel filtration of the components indicates an ap- parent molecular weight of 58,000. The 3.1S binding mole- tine in a manner similar to the binding of steroid cules are not observed in adrenal , testes, liver, or kid- to their respective target organs. 1a,25-(OH)2D3 enters the ney, but similar receptors for la,25-(0H)2D3 have been found intestinal cell and binds to a 3.7S cytoplasmic receptor pro- previously in intestine. tein (17, 18). The hormone receptor complex then migrates Macromolecular species with a high affinity and prefer- into the nucleus in a temperature-dependent process, where ence for 25-hydroxyvitamin D3 [25(0H)D3] are also identi- it associates with the chromatin (15, 17-19). We report here fied in parathyroid cytosol and differ from the parathyroid similar intracellular receptor proteins for la,25- la,25(OH)WD3-binding component in that: (1) they sediment that at 6 S in 0.3 M KCI-sucrose gradients, (2) they are observed in (OH)2D3 exist in chick parathyroid glands. all tissues examined, (3) they have a higher affinity for 25- (OH)D3 than la,25-(0H)2D3, and (4) they are not found in the MATERIALS AND METHODS- nucleus of the parathyroid glands, in vitro. The discovery of unique la,25.OH)2D3-binding components in the parathy- Materials. Animals used in experiments were White Leg- roid glands is consistent with the sterol hormone's action at horn cockerels (kindly donated by Demler Farms, Anaheim, this endocrine site and possible involvement in the regula- Calif.) that were raised for 6 weeks on a vitamin-D-deficient tion of synthesis and secretion. diet (20). 25-Hydroxy[26(27)-methyl-3H]vitamin D3 (6.5 Ci/ mmol) was obtained from Amersham-Searle. Vitamin D3 action to mobilize and phosphate at in- Preparation of la,25-Dihydroxy[3Hjvitamin D3, In testine and bone is thought to be mediated by the metabolite Vitro. la,25-Dihydroxy[26(g7)-methyl-3H]vitamin D3 was la,25-dihydroxyvitamin D3 [la,25-(OH)2D3] (1-4). Its pro- prepared as previously described (19). Radiochemical purity duction from 25-hydroxyvitamin D3 [25-(OH)D3] by the of generated la,25-dihydroxy[3H]vitamin D3 was 98%. 25- renal la-hydroxylase appears to be regulated by calcium (5), Hydroxy[26(27)-methyl-3H]vitamin D3 substrate for the re- phosphate (6, 7), parathyroid hormone (PTH) (7, 8), and the action was purified by Celite liquid-liquid partition chro- vitamin D status of the animal (9). Evidence suggests that matography (21). The radiochemical purity of the 25-hy- stimulates PTH secretion which in turn en- droxy[3H]vitamin D3 was 95%, and its specific activity was hances the production of la,25-(OH)2D3 at the (7, determined by ultraviolet absorbance spectrophotometry at 8). Thus PTH, rather than calcium, may be the dominant 265 nm. modulator of the renal la-hydroxylase (10) and the finding Exposure of Chick Tissue Subfractions to Radioactive of abnormal circulating 1a,25-(OH)2D3 in humans with Sterols, In Vitro. Homogenates [300 mg wet weight (20 is consistent with this concept (11, 12). parathyroid glands)/3 ml] were made in 0.25 M sucrose, DeLuca has proposed that PTH and phosphate deficiency 0.05 M Tris-HCI, pH 7.4, 0.025 M KCI, and 5 mM MgC12 may be functioning through a common intracellular mecha- (0.25 M sucrose-buffer A) with a Potter-Elvehjem homoge- nism to enhance the la-hydroxylase by lowering the phos- nizer equipped with a Teflon pestle at 00 by six passes, with phate level in the renal cell (10). Moreover, MacIntyre and 2 min cooling periods between passes. Homogenates were associates (13) have suggested that la,25-(OH)2D3 might centrifuged at 1200 X g for 10 min. Nuclear pellets were re- control its own biosynthesis directly at the kidney by a nega- moved, and the resulting supernatant was centrifuged at tive feedback mechanism involving the de novo synthesis of 100,000 X g for 1 hr at 0° to yield a final supernatant frac- the la-hydroxylase enzyme. Clearly, the fashion in which tion (cytosol). The cytosol (0.2-1.0 ml) was incubated with la,25-(OH)2D3, PTH, and phosphate deficiency interact to sterol (in 20,l ethanol) for 1 hr at 00 and then analyzed for control the formation of 1a,25-(OH)2D3 at its kidney endo- sterol binding components. crine site must be completely understood before the exact Purified nuclear extracts (chromatin) were prepared from nuclear pellets by a modification (7) of the method of Haus- Abbreviations: 25-(OH)D3, 25-hydroxyvitamin D3; la-(OH)D3, sler et al. (1) and were resuspended in 0.01 M Tris-HCl, pH la-hydroxyvitamin D3; la,25-(OH)2D3, la,25-dihydroxyvitamin 7.5, and centrifuged for 20. min at 48,000 X g. The pellet D3; PTH, parathyroid hormone. from 300 mg of tissue was reconstituted with cytosol (2.5 ml) 4871 Downloaded by guest on October 1, 2021 4872 Biochemistry: Brumbaugh et al. Proc. Nat. Acad. Sci. USA 72 (1975)

and incubated for 1 hr at 25° with sterol (in 40 Al of etha- nol). Chromatin was harvested and extracted with 0.3 M KC1, 0.01 M Tris-HCl, pH 7.5, 1.5 mM EDTA, 12 mM 1- thioglycerol (0.3 M KCl-Buffer B). Extracts were centri- fuged at 48,000 X g for 20 min, and the resulting superna- tants were analyzed for sterol-binding activity. Sucrose Gradient Centrifugation. Linear gradients (5.0 ml) of 5-20% sucrose in 0.3 M KCI-Buffer B were prepared with a Buchler gradient mixer, Auto-Densi Flow, and Poly- staltic pump. Aliquots (0.3 ml) of cytosol or nuclear extracts were layered on gradients and centrifuged at 234,000 X g (average force) for 24 hr at 00 with the use of a Beckman L3-50 ultracentrifuge and an SW 50.1 rotor. The fractions (6 drops each) were counted in 5 ml of liquid scintillation mix- ture A (3% Liquifluor in toluene-Triton X-114, 3:1) in a Beckman LS-233 scintillation counter (35% efficiency). Sedi- mentation coefficients were estimated by comparison with protein markers (chymotrypsinogen, 2.5 S; ovalbumin, 3.67 S; and bovine serum albumin, 4.4 S). Agarose Gel Filtration. All chromatographic procedures were carried out at 1-3'. Agarose beads (Bio-Gel A-0.5m, 100 to 200 mesh from Bio-Rad) were equilibrated with 0.3 M KCI-Buffer B and poured into a column (1.6 X 60 cm). Samples (1.0 ml) of nuclear extracts or cytosol incubations were applied to the column and 1-ml fractions were collect- ed and counted (30% efficiency). The optical density of 20 TOP 10 fractions was measured with a Gilford 240 spectrophotome- FRACTION NUMBER ter. Column flow rates were maintained at 13-14 ml/hr FIG. 1. Sucrose gradient centrifugation of la,25-(OH)2D3 and with a Polystaltic pump. 25-(OH)D3-binding components in parathyroid glands and nontar- Filter Assay for Specific Macromolecular Binding. Sep- get organs. Incubations were carried out as follows: (A) Parathy- aration of bound from free sterol was achieved the filter roid cytosol (0.3 ml) at 00 for 1 hr with 3 nM la,25-(OH)2[3H]D3 by (6.5 Ci/mmol) alone (0), or with 0.3 gM unlabeled la,25-(OH)2D3 assay method of Santi et al. (22). Aliquots of cytosol (0.2 ml) (0), or with 6 nM nonradioactive 25-(OH)D3 (A). (B) Reconsti- containing la,25(OH)2[3H]D3 and samples containing the tuted cytosol-chromatin from parathyroid (@), adrenal (A), or same concentration of 1a,25-(OH)2[3H]Ds plus a 100-fold testes (A) was incubated with 6 nM la,25-(OH)2[3H]D3 for 1 hr at excess of unlabeled hormone were incubated at 00 for 2 hr. 250. The chromatin was then extracted with 0.3 M KCl-Buffer B. was then to DEAE-cellulose filters Parallel incubation with parathyroid cytosol-chromatin, 6 nM Cytosol (150 Al) applied lz,25-(OH)2[3H1D3, and 0.6 'M unlabeled la,25-(OH)2D3 was per- (Whatman DE 81) and washed with three 1-ml portions of formed (0). (C) Parathyroid cytosol with 6 nM 25-(OH)[3H]D3 (6.5 1% Triton X-100 in 0.01 M Tris-HCI, pH 7.5, with the use of Ci/mmol) alone (0), or with 60 nM nonradioactive 25-(OH)D3 (O). a Millipore sampling manifold. The amount of la,25- or with 60 nM unlabeled la,25-(OH)2D3 (A). (D) Testes cytosol (OH)2[3H]D3 specifically bound by the cytosol was deter- with 6 nM 25-(OH)[3H]D3 (0); testes cytosol with 6 nM la,25- mined as previously described (17). Efficiency of the filtra- (OH)2[3H]D3 alone (A) or with 0.6 MM unlabeled la,25-(OH)2D3 tion as determined measurement of (A) or with 60 nM nonradioactive 25-(OH)D3 (0). Arrows indicate procedure, by binding sedimentation of standards: at low hormone was 75%. positions protein 1, chymotrypsino- concentrations, gen; 2, ovalbumin; 3, bovine serum albumin. RESULTS Initially, parathyroid gland was studied to determine if it tively compete with 1a,25-(OH)2D3 (see Table 1). Since the contained a soluble binding component for the Ila,25- shoulder disappeared in the presence of 25-(OH)D3 and cor- (OH)2D3 hormone. Parathyroid cytosol from vitamin-D-de- responded to the sedimentation position (6S) of the high-af- ficient chicks was incubated at 0° with ia,25-(OH)2[3H]Da and binding of the sterol to macromolecules was analyzed Table 1. Influence of unlabeled sterols on the binding of by sucrose gradient centrifugation. As depicted in Fig. 1A, labeled hormone to cytoplasmic parathyroid the radioactive hormone interacted with a macromolecular la,25-(OH)2D3-binding component species sedimenting at 3.1 S. Parallel cytosol incubations containing excess unlabeled 1a,25-(OH)2D3 showed no ra- Unlabeled sterol dioactive hormone binding in the sedimentation position of (concentration, nM) % Binding the 3.1S-binding component (Fig. 1A), demonstrating that the interaction of hormone with this cytosol molecule is satu- None 100 rable and of high affinity. Sucrose gradients of the la,25- 1a,25-(OH)2D3 (400) 0 (OH)2D3-binding component in parathyroid cytosol repro- 1,25-(OH)2D3 (4) 53 55 ducibly exhibited a faster-sedimenting shoulder on the pri- 25-(OH)D3 (80) 95 mary peak. When a 2-fold excess of nonradioactive 25- la-(OH)D3 (2,000) (OH)D3 was included in the incubation (Fig. 1A), the shoul- Vitamin D3 (10,000) 98 der was abolished without detectable effect on any la,25- Aliquots of cytosol were incubated with 4 nM la,25-(OH)2- (OH)2[3H]Dj binding to the 3.1S macromolecule. Thus, this [3H]D3 and unlabeled sterol for 1 hr at 00. Binding of radioactive macromolecule selectively binds the hormone and much sterol to the 3.1S component was determined via sucrose gradient higher concentrations of 25-(OH)D3 are required to effec- centrifugation. Downloaded by guest on October 1, 2021 Biochemistry: Brumbaugh et al. Proc. Nat. Acad. Sci. USA 72 (1975) 4873

-J 0II -J 0 (I) 0

-jz z ) 0 0~H z ) C) U- LL N v4 I) I I

FRACTION NUMBER FIG. 2. Agarose gel filtration of cytoplasmic and nuclear la,25-(OH)2D3-binding components of chick parathyroid glands. Reconstituted cytosol-chromatin (1 ml) was incubated with la,25-(OH)2[3H]D3 (4 nM) for 1 hr at 250. The chromatin was then extracted with 0.3 M KC1- Buffer B (0-*). One ml of parathyroid cytosol (prepared in 0.3 M KCl-Buffer B) was incubated with la,25-(OH)2[3HJD3 (6 nM) for 1 hr at 00 (0-0). (-), relative absorbance at 280 nm of cytosol eluate. Molecular weights of the components were estimated from a linear plot of Mr112 versus the distribution coefficient, KD1 3, with the use of myoglobin, chymotrypsinogen, pepsin, ovalbumin, and bovine serum albumin as standards. Vo and Vt are the void and total volumes, respectively.

finity binding protein for 25-(OH)D3 discovered by Haddad cytosol-chromatin at 250 for 1 hr and the chromatin was and Birge in the rat (23), it was concluded that this shoulder then harvested and extracted with 0.3 M KCI-Buffer B. Su- represented association of the hormone with this 6S compo- crose gradient analysis of this nuclear extract resulted in a nent. 3.1S peak of bound 1a,25-(OH)2[3H]Da (Fig. 1B). Parallel To further investigate the interrelationship of the 3.1S incubations containing a 100-fold excess of unlabeled hor- binding component and the 6S species which binds both mone showed a striking reduction in the radioactive sterol 1a,25-(OH)2D3 and 25-(OH)D3, parathyroid cytosol was in- bound to this macromolecule. Also, the association of la,25- cubated with radioactive 25-(OH)D3. Sucrose gradient anal- (OH)2[3H]D3 with a nuclear component was not observed in ysis (Fig. IC) indicates that 25-(OH)[3H]D3 binds exclusive- the adrenals or testes (Fig. 1B). Therefore, the parathyroid ly to a protein sedimenting at 6 S. Parallel incubations con- nuclear chromatin contains a tissue-specific, high-affinity taining a 10-fold excess of either nonradioactive 25-(OH)D3 binding component for 1a,25-(OH)2D3 which is indistin- or la,25-(OH)2D3 show that this protein has a higher affini- guishable from the cytosol macromolecular species by su- ty for 25-(OH)D3 than for the hormone; binding to the 6S crose gradient centrifugation. peak was reduced by 15% in the presence of 1a,25-(OH)2D3 The cytoplasmic and nuclear la,25-(OH)2D3-binding and by about 90% in the presence of 25-(OH)D3 (Fig. IC). components were also isolated and compared by agarose gel The 25-(OH)D3-binding component in parathyroid cytosol filtration chromatography in 0.3 M KCI-Buffer B (Fig. 2). is therefore similar to proteins found in a variety of rat The major peak represents [3H]sterol binding to a macro- tissues by Haddad and Birge (23) and to proteins reported in molecule of apparent molecular weight 58,000, which is re- chick intestine, liver, and kidney by Brumbaugh and Haus- solved from the major protein peak (eluting in the void vol- sler (24). In all cases, the binding component has a greater ume). No significant or reproducible difference could be affinity for 25-(OH)D3 than 1a,25-(OH)2D3 and sediments demonstrated between the nuclear and cytoplasmic compo- at 6 S in sucrose gradients. nents when columns were run under identical, standardized Analysis of la,25-(OH)2[3H]Da interactions with other tis- conditions (Fig. 2) and both peaks of macromolecule- sue cytosols showed that the 3. IS hormone-binding compo- [3H]hormone complexes were abolished when incubations nent exists only in parathyroid gland and is not present in containing excess unlabeled la,25-(OH)2D3 were chromato- testes (Fig. ID), , liver, or kidney (data not graphed (data not shown). Thus, the specific nuclear and cy- shown). Detailed investigation of testes cytosol (Fig. ID) re- toplasmic 1a,25-(OH)2D3-binding components could not be vealed that the la,25-(OH)2D3 hormone associates predomi- distinguished from each other by the ultracentrifugal and nantly with the 25-(OH)Ds-binding protein (sedimenting at chromatographic techniques employed. These data are con- 6 S). However, the preference of this protein for 25-(OH)D3 sistent with the concept that the nuclear component origi- is demonstrated by the greater potency of unlabeled 25- nates in the cytosol and suggest that 1a,25-(OH)2D3 may be (OH)D3 over la,25-(OH)2D3 in abolishing this 6S peak (Fig. functioning in the parathyroid gland at the level of the cell iD). A small amount of 1a,25-(OH)2[3H]D3 binds to mole- nucleus. cules in testes cytosol sedimenting at 3-4 S, but this binding Next the binding affinity and specificity of the cyto- is low affinity and nonspecific because it is not reduced by plasmic-binding components for 1a,25-(OH)2D3 in parathy- excess unlabeled Ia,25-(OH)2D3. Therefore, with the excep- roid glands were studied. Incubation of parathyroid cytosol tion of the target intestine, where a selective 3.7S hormone with increasing concentrations of 1a,25-(OH)2[3H]D3 and receptor is found (18), cytosol components specific for the determination of specific binding by the filter method of la,25-(OH)2D3 hormone over its 25-(OH)D3 precursor are Santi et al. (22) showed the saturation of a limited number detected only in parathyroid glands. of binding sites (Fig. 3A). Saturation occurs at low concen- The association of la,25-(OH)2D3 with nuclear-binding trations of hormone (3 X 10-9 M). At this range of hormone components from parathyroid gland was also observed in concentration most of the binding detected by the filter vitro. la,25-(OH)2[3H]D3 was incubated with reconstituted assay is specific for la,25-(OH)2D3, since only a small Downloaded by guest on October 1, 2021 4874 Biochemistry: Brumbaugh et al. Proc. Nat. Acad. Sci. USA 72 (1975)

FIG. 3. Determination of dissociation constant for la,25-(OH)2D3-parathyroid cytosol macromolecule interaction. (A) Specific binding. of la,25-(OH)2[3H]D3 by parathyroid cytosol. Aliquots of cytosol (0.2 ml, 2.0 mg of protein per ml) were incubated with increasing amounts of la,25-(OH)2[3H]D3 in the presence (nonspecific) or absence (total) of a 100-fold excess of unlabeled la,25-(OH)2D3 for 2 hr at 00. (B) Scatchard analysis of specific binding in A. Three determinations indicate that the 1a,25-(OH)2[3H]D3 macromolecule interaction has a Kd = 3.2 4 0.2 X 10-10 M at 00, where the uncertainty is expressed as standard deviation.

amount of labeled hormone is bound in the presence of a protein has a higher affinity for 25-(OH)D3 than the hor- 100-fold excess of unlabeled hormone (nonspecific binding). mone, it will also bind la,25-(OH)2[3H]Ds when incubated Scatchard analysis of the specific binding (total minus non- with the sterol at -10-8 M in vitro (Fig. IA and D, Table 2). specific) is linear (Fig. 3B), suggesting a single class of bind- However, in the target intestine and in the parathyroid ing sites. The dissociation constant for the hormone-macro- gland, unique macromolecules sedimenting at 3.7 S and 3.1 molecule complex at 00 is 3.2 X 10-10 M. S, respectively, are observed to selectively bind la,25- Analogs of la,25-(OH)-D3 which lack a hydroxyl group at (OH)2D3 and transfer the hormone into the cell nucleus. the la- and/or 25-position were tested for their ability to These binding components are analogous to the classic ste- compete with the labeled hormone for binding to the 3.1S roid hormone receptors and, based upon the pattern of bind- cytoplasmic component. As is shown in Table 1, the la,25- ing proteins established in Table 2, the parathyroid gland (OH)2D3-binding macromolecule is highly specific. The ap- proximate relative affinity of these sterols for the 3.1S mac- romolecule is 1:1/20:<1/500:<1/2500 for la,25-(OH)2D3: Table 2. Sedimentation coefficients of high-affinity 25-(OH)Ds:la-(OH)D3:vitamin D3. These values differ binding proteins for 25-(OH)D3 and 1a,25-(OH)2D3 from the 1:1/500:1/800:<1/20,000 relative affinities for in the chick* these sterols' association with the 3.7S receptor of intestine (25). The present results indicate that the parathyroid gland Sedimentation differs from the well- coefficient, receptor for la,25-(OH)2D3 slightly S characterized hormone receptor in the intestine in terms of (±SD) sedimentation coefficient, affinity, and specificity. Yet it is Sterol Site Cytosol Nuclear Refs. clear that the parathyroid gland should be included with the intestine as a location of specific, high-affinity binding com- 25-(OH)D3 Intestine 6 t 24 ponents for the la,25-(OH)2D3 hormone. Parathyroid 6 t Testes 6 t DISCUSSION Liver 6 t 24 Kidney 6 t 24 The existence of a specific la,25-(OH)2D3-binding macro- Serum 4.0 - 24 molecule has been demonstrated in chick parathyroid ± 0.1 is glands. Although the function of this binding component lc,25-(OH)2D3 Intestine 3.7 3.7 17, 18 not known, it has been definitively distinguished from the ± 0.1 ±0.1 25-(OH)D3-binding protein which has been found in the rat Parathyroid 3.1 3.1 and chick (23, 24). Table 2 summarizes data on the proper- ± 0.2¶ + 0.2¶ ties of chick vitamin D metabolite-binding proteins in terms Testes 6 § t t of their sedimentation coefficients and subcellular location. Liver 6 § t 26 A 6S 25-(OH)Ds-binding component has been identified in Kidney 6 § t 26 the cytosol of all organs examined in the chick, but this pro- Serum 4.0 - 24 tein is not found in the nucleus. At present, a role for this ± 0.1¶ macromolecule has not been found, but the fact that it does not transport 25-(OH)D3 into the nucleus suggests that it is * Data from 0.3 M KCl-sucrose gradients. not a classic receptor (27, 28). The tissue t Not observed. from t Present study. 25-(OH)Ds-binding protein can also be differentiated § Represents binding of la,25-(OH)2[3H]D3 to 25-(OH)D3 binding the serum protein which binds either 25-(OH)D3 or la,25- protein. (OH)2DW and sediments at 4.0 S in sucrose gradients (Table Average of four sucrose gradient experiments; significantly dif- 2). We have also observed that although the ubiquitous 6S ferent from 3.7S intestinal receptor (P < 0.005). Downloaded by guest on October 1, 2021 Biochemistry: Brumbaugh et al. Proc. Nat. Acad. Scd. USA 72 (1975) 4875 LOW CALCIUM relationship should further our understanding of both the normal physiology of these ions and of diseases of mineral metabolism such as primary , idiopath- PARATHYROID CELL ic hypercalciuria, and vitamin D-resistant rickets. PTH NUCLEUS We gratefully acknowledge the expert technical assistance of Ms. I Kristine Bursac and Ms. Patricia G. Jones. The authors wish to R ANDSE(-C Xl2 thank the National Institutes of Health, Grant AM-15781-04 and Training Grant GM-01982, for support of this research. 0] (§)-1,25 ALCIUM RETENTION \ I 1. Haussler, M. R., Myrtle, J. F. & Norman, A. W. (1968) J. Biol. PTH 1 HOSPHATE EXCRETION Chem. 243,4055-4064. 2. Fraser, D. R. & Kodicek, E. (1970) Nature 228,764-766. RENAL lIc-OHase (CALCII JM / 3. Haussler, M. R., Boyce, D. W., Littledike, E. T. & Rasmussen, 25-OH-D3 --- I,25-(OH( 3 APHOSF) H. (1971) Proc. Nat. Acad. Sci. USA 68, 177-181. PHATE - I 4. Holick, M. F., Schnoes, H. K., DeLuca, H. F., Suda, T. & tMUUIMDib LlLI IZATION Cousins, R. J. (1971) Biochemistry 10,2799-2804. - HS - 5. Boyle, I. T., Gray, R. W. & DeLuca, H. F. (1971) Proc. Nat. LOW PHOSPHATE Acad. Sci. USA 68,1231-2134. 6. Tanaka, Y. & DeLuca, H. F. (1973) Arch. Biochem. Biophys. FIG. 4. Integration of possible 1a,25-(OH)2D3 action at para- 154,566-574. gland with the homeostatic regulation of serum calcium M. P. M. and phosphate by the concerted functioning of PTH and la,25- 7. Hughes, R., Brumbaugh, F., Haussler, R., Wergedal, (OH)2D3. J. E. & Baylink, D. J. (1975) Science 190,578-50. 8. Garabedian, M., Holick, M. F., DeLuca, H. F. & Boyle, I. T. (1972) Proc. Nat. Acad. Sci. USA 69, 1673-1676. 9. Tucker, G., Gagnon, R. E. & Haussler, M. R. (1973) Arch. Bio- should perhaps be classified along with the intestine and chem. Biophys. 155,47-57. bone as a target site for 1a,25-(OH)2D3. 10. DeLuca, H. F. (1974) Fed. Proc. 33, 2211-2219. Little information was available until recently on the pos- 11. Brumbaugh, P. F., Haussler, D. H., Bressler, R. & Haussler, M. sible function of la,25-(OH)2D3 at the parathyroid gland. R. (1974) Science 183, 1089-1091. Oldham et al. (29) have detected and characterized a cal- 12. Haussler, M. R., Bursac, K. M., Bone, H. & Pak, C. Y. C. cium-binding protein in parathyroid gland which is similar (1975) Clin. Res. 23, 322A. to the intestinal calcium-binding protein induced by la,25- 13. Larkins, R. G., MacAuley S. J. & MacIntyre, I. (1974) Nature 252,412-414. It is that the of serum cal- (OH)2D3 (30, 31). possible sensing 14. Henry, H. L. & Norman, A. W. (1975) Biochem. Biophys. cium by the parathyroids is dependent upon 1a,25-(OH)2D3 Res. Commun. 62,781-788. and the synthesis of a calcium-binding protein in this gland. 15. Brumbaugh, P. F. & Haussler, M. R. (1973) Biochem. Bio- Also, Chertow et al. (32) have reported that la,25-(OH)2D3 phys. Res. Commun. 51, 74-80. suppresses PTH secretion in the rat, in vvo, and in isolated 16. Tsai, H. C. & Norman, A. W. (1973) J. Biol. Chem. 248, slices of bovine parathyroid gland. These observations 5967-5975. suggest that 1a,25-(OH)2D3, like other steroid hormones 17. Brumbaugh, P. F. & Haussler, M. R. (1974) J. Biol. Chem. (33), feedback inhibits its trophic counterpart, PTH. A 249, 1258-1262. model depicting the way in which this feedback loop may 18. Brumbaugh, P. F. & Haussler, M. R. (1975) J. Biol. Chem. integrate into the complex scheme for the regulation of cal- 250, 1588-1594. 19. Brumbaugh, P. F. & Haussler, M. R. (1974) J. Biol. Chem. cium and phosphate is illustrated in Fig. 4. The two primary 249, 1251-1257. signals in this are postulated to be low cir- 20. McNutt, K. M. & Haussler, M. R. (1973) J. Nutr. 103, 681- culating calcium and low phosphate (7, 10). Low calcium 689. enhances the formation of 1a,25-(OH)2D3 via a stimulation 21. Haussler, M. R. (1972) Steroids 20,639-650. of parathyroid hormone secretion (7, 8). The calcium mobi- 22. Santi, D. V., Sibley, C. H., Perriard, E. R., Tomkins, G. M. & lized from bone by the synergistic action of PTH and Baxter, J. D. (1973) Biochemistry 12, 2412-2416. 1a,25-(OH)2D3 and that absorbed from intestine under the 23. Haddad, J. G. & Birge, S. J. (1975) J. Biol. Chem. 250, 299- influence of enhanced sterol levels probably closes the hor- 303. mone loop by abolishing further PTH secretion (34). Direct 24. Brumbaugh, P. F. & Haussler, M. R. (1975) Life Sci. 16, feedback of at the parathyroid gland may 353-362. 1a,25-(OH)2D3 25. Haussler, M. R. & Brumbaugh, P. F. (1976) in Molecular As- also be involved in the regulation of PTH secretion during pects of Hormone-Receptor Interaction, ed. Levey, G. S. the correction of low serum calcium. (Marcel Dekker, Inc., New York), in press. The significance of the operation of 1a,25-(OH)2D3 at the 26. Brumbaugh, P. F. (1975) Doctoral Dissertation, University of parathyroid gland may be more profound in terms of phos- Arizona. phate . During phosphate depletion the kidney is 27. O'Malley, B. W. & Means, A. R. (1974) Science 183,610-620. stimulated to form more 1a,25-(OH)2D3 (Fig. 4; refs. 6 and 28. Edelman, I. S. (1975) J. Steroid Biochem. 6, 147-159. 7). To maintain the phosphate mobilized by the sterol, PTH 29. Oldham, S. B., Fischer, J. A., Shen, L. H. & Arnaud, C. D. secretion must be curtailed (because PTH causes net phos- (1974) Biochemistry 13, 4790-4796. phate excretion). Such curtailment can ultimately occur 30. Wasserman, R. H. & Taylor, A. N. (1968) J. Biol. Chem. 243, after is action la,25- 3987-3993. hypercalcemia established by the of 31. Corradino, R. A. (1973) Nature 243,41-43. (OH)2D3, but a more plausible mechanism would be the di- 32. Chertow, B. S., Baylink, D. J., Wergedal, J. E., Su. M. H. H. & rect inhibition of PTH secretion by 1a,25-(OH)2D3 (Fig. 4). Norman, A. W. (1975) J. CGn. Invest. 56,668-678. The control of calcium and phosphate is apparently accom- 33. Thomas, P. J. (1973) J. Endocrinol. 57,333-359. plished by a complex and delicate interrelationship between 34. Arnaud, C. D., Tenenhouse, A. M. & Rasmussen, H. (1967) PTH and 1a,25-(OH)2D3 and a comprehension of this exact Annu. Rev. Physiol. 29,349-372. Downloaded by guest on October 1, 2021