Parathyroid hormone gene expression in
hypophosphatemic rats.
R Kilav, … , J Silver, T Naveh-Many
J Clin Invest.
1995;
96(1)
:327-333.
https://doi.org/10.1172/JCI118038
.
Phosphate is central to bone metabolism and we have therefore studied whether
parathyroid hormone (PTH) is regulated by dietary phosphate in vivo. Weanling rats were
fed diets with different phosphate contents for 3 wk: low phosphate (0.02%), normal calcium
(0.6%), normal phosphate (0.3%), and calcium (0.6%); high phosphate (1.2%), high calcium
(1.2%). The low phosphate diet led to hypophosphatemia, hypercalcemia, and increased
serum 1,25(OH)2D3 together with decreased PTH mRNA levels (25 +/- 8% of controls, P <
0.01) and serum immunoreactive PTH (4.7 +/- 0.8: 22.1 +/- 3.7 pg/ml; low phosphate:
control, P < 0.05). A high phosphate diet led to increased PTH mRNA levels. In situ
hybridization showed that hypophosphatemia decreased PTH mRNA in all the parathyroid
cells. To separate the effect of low phosphate from changes in calcium and vitamin D rats
were fed diets to maintain them as vitamin D-deficient and normocalcemic despite the
hypophosphatemia. Hypophosphatemic, normocalemic rats with normal serum
1,25(OH)2D3 levels still had decreased PTH mRNAs. Nuclear transcript run-ons showed
that the effect of low phosphate was posttranscriptional. Calcium and 1,25(OH)2D3 regulate
the parathyroid and we now show that dietary phosphate also regulates the parathyroid by a
mechanism which remains to be defined.
Research Article
Find the latest version:
Parathyroid Hormone
Gene Expression
in
Hypophosphatemic
Rats
RachelKilav, Justin Silver, and Tally Naveh-ManyMinervaCenterfor Calcium and BoneMetabolism, Nephrology Services, Hadassah University Hospital, Jerusalemil-91120,Israel
Abstract
Phosphate is centraltobone metabolism andwehave there-fore studied whether parathyroidhormone (PTH)is
regu-lated by dietary phosphate in vivo. Weanling rats were
fed diets with different phosphate contents for 3 wk: low
phosphate (0.02%),normal calcium(0.6%),normal phos-phate(0.3%), and calcium (0.6%); high phosphate (1.2%), high calcium (1.2%). The low phosphate dietled to hypo-phosphatemia, hypercalcemia, and increased serum
1,25(OH)2D3 together with decreased PTH mRNA levels
(25±8% of controls, P< 0.01) andserumimmunoreactive
PTH (4.7±0.8: 22.1±3.7 pg/ml; low phosphate: control,P
<0.05). A high phosphatediet led to increased PTH mRNA
levels. In situ hybridization showed that hypophosphatemia decreasedPTH mRNA in all theparathyroidcells. To
sepa-rate the effect of low phosphate from changes in calcium andvitamin Dratswerefeddiets tomaintain themas vita-minD-deficient and normocalcemic despite the hypophos-phatemia. Hypophosphatemic, normocalemicratswith
nor-mal serum 1,25(OH)2D3 levels still had decreased PTH
mRNAs. Nuclear transcriptrun-onsshowedthat the effect
of low phosphate was posttranscriptional. Calcium and
1,25(OH)2D3 regulate the parathyroid and we now show thatdietary phosphate alsoregulates the parathyroid bya
mechanism which remains to be defined. (J. Clin. Invest 1995.96:327-333.) Key words: parathyroid cells *calcium * 1,25-dihydroxyvitamin D3 * secondary
hyperparathyroid-ism *phosphate* parathyroid hormone
Introduction
Parathyroid hormone (PTH)' geneexpression is known to be regulated by 1,25-dihydroxyvitamin D3 (1,25 (OH)2D3),
cal-The results of thisstudywerepresentedinpart attheAmericanSociety of Bone and MineralResearch, Tampa, FL, 18-22September 1993, KansasCity, MO,9-13September 1994, andwerepublished in abstract
form(1993. J. Bone Miner. Res. 8:S200; 1994. J. Bone Miner. Res. 9:S338).
AddresscorrespondencetoJustinSilver, MineralMetabolism Unit, HebrewUniversitySchool ofMedicine,HadassahUniversity Hospital, P.O. Box 12000, 91120, Jerusalem,Israel. Phone: 011-972-2436-778; FAX:011-972-2421-234.
Receivedfor publication7November 1994 and accepted in revised form27February1995.
1.Abbreviations usedinthispaper:1,25 (OH)2D3,
1,25-dihydroxyvita-min D3; iPTH,immunoreactiveparathyroidhormone; PTH, parathyroid hormone; VDR, vitamin Dreceptor.
cium, and estrogens (1). 1,25(OH)2D3 decreases PTH gene
transcription both in vivo in rats (2) and in vitro in primary cultures ofbovine parathyroid cells (3), aswellasinastably transfected cell line (4, 5). In vivo in rats hypocalcemia
in-creases PTH mRNA levels (6-8). Estrogens also lead to an
increase in PTH mRNA levels (9).
Hyperphosphatemia leads to adecrease in serum calcium and1,25(OH)2D3 levels,andhypophosphatemiato anincrease inserum1,25 (OH)2D3 levels (10). Because of the importance of thephosphate retention of chronicrenal failure to the patho-genesis ofsecondary hyperparathyroidism,aswell asthe
impor-tance ofphosphatetonormalphysiology,wehave now studied
the effects of phosphate on PTH gene expression in vivo in
rats.Ourpresentstudy shows thatchanges in dietary phosphate regulate PTHgeneexpression andserum PTHlevels.
Methods
Animals.Weanling male HebrewUniversity strainratswere maintained for 3 wk on the following diets (Teklad Premier Laboratory Diets, Madison, WI): Low phosphate, normal calcium (0.02% phosphate, 0.6% calcium); normal phosphate, normalcalcium (0.3% phosphate, 0.6% calcium); high phosphate, high calcium (1.2% phosphate, 1.2% calcium); vitaminD-deficient, low phosphate, low calcium (0.02% phosphate, 0.02% calcium). After 1-21 d the thyroparathyroid tissue wasexcised underpentobarbital anesthesia, and blood samples taken. Allratsurgery wasperformed at9:00-10:00 a.m.The excisedtissue was immediately frozen inliquid nitrogen and stored at -700Cuntil extraction.
Second generationvitaminD-deficientrats.Femalerats were main-tainedonvitamin D-free diets from weaning until maturity and their offspring fed diets with no vitamin D and low or normal phosphate concentrations (as above). In another experiment second generation vitaminD-deficient rats werefed a vitamin D-deficient diet low in bothphosphate and calcium (as above).
Measurementof cellular mRNA levels. RNA was extracted from rat thyroparathyroid tissue and the levels ofPTHmRNA weremeasured by Northern blots after extraction with TRI Reagent (Molecular Research Center, Inc., Cincinnati OH). RNAwasdenatured and ethidium bromide wasaddedtoeach sampleat aconcentration of0.1 mg/ml. The samples weresize-fractionated by electrophoresis on 1.25% agarose gels con-tainingformaldehyde and transferredtoHybond filters(Amersham In-ternational, Little Chalfont, United Kingdom) by diffusion blotting. The integrityoftheRNA andtheuniformity of RNA transfer to the mem-brane were determined by ultraviolet visualization of the ribosomal RNAbands of the gels andthefilters. The filters were fixed by ultraviolet cross-linking and hybridizedaspreviously described (2, 9). Hybridiza-tion was to a randomprimed rat PTH cDNA (a gift of H. Meyer, Gesellschaft fur Biotechnologische Forschung, Braunschweig, Ger-many)and 18SRNA(giftof M. A. Levine,JohnsHopkinsHospital, Baltimore, MD).
Nuclear run-on transcriptionassay. Thyroparathyroid tissue from 10 rats washomogenized with apolytronin0.25 M sucrose, 10 mM Hepes pH 0.8, 10mM MgCl2,2 mM DTT, and 0.1% TritonX-100 (homogenization buffer). Nuclei werepelletedat 500g at4°C for 5 min, thenresuspendedinhomogenization buffer. The nuclearsuspension waslayeredover a sucrose cushionconsistingof1 Msucrose, 10 mM Hepes (pH 8.0), 10mMMgCl2,2 mMDTT,and0.1%TritonX-100,
J.Clin. Invest.
© TheAmericanSociety for Clinical Investigation, Inc. 0021-9738/95/07/0327/07 $2.00
TableI. TheEffects of 3 wk of Diets Given to Weanling Rats
Serum phosphate Serum calcium Serum1,25(OH)2D3
(meantSE [n]) (meantSE[n]) (meantSE [n]) mg/dl mg/dl pg/mi
Low phosphate,
normal calcium 4.0±0.4 (4)t 12.6±0.6 (4)* >400(4)t
Normalphosphate,
normal calcium 9.8±1.2 (4) 10.6±0.4 (4) 68±28.3 (4) Highphosphate,
high calcium 9.4±0.8 (4) 11.1±0.6 (4) 30±5.9 (4)* n, number ofrats. * P <0.05,or *P <0.01 compared with normal
phosphate, normal calcium.
and pelleted at 500 g at 4°C for 5 min. Nuclei were resuspended in 40% glycerol, 5 mM MgCl2, 50 mM Tris, and 0.1 mM EDTA (storage buffer), then frozen in liquid nitrogen, and storedat-70°C (11). For therun-onassay( 12) nucleiwerethawedonice, then incubated in
200-ttl
reaction mixture containing 30% glycerol, 2.5 mM DTT, 1 mMMgCl2, 70mMKCI, 0.5 mMeachofATP,CTP, and GTP, 1 mg/ml heparin, 100 ,uCi of[32p]UTP(800 ci/mmol), and 100URNase inhibi-tor, for 20 minat26°C. Nuclear-labeled RNAwasextracted withTRI Reagent(Molecular Research Center, Inc).RNA waspelleted and re-suspended in200-,LIhybridization buffer (7% SDS, 10% polyethylene glycol (8,000), 1.5 xsodium chloride sodium phosphate EDTA). Ali-quots of RNA from treated and untreated sampleswerecounted in a scintillation counter, andanequalnumber ofcountsfrom each condition
(1-2x106 cpm)washybridizedto cDNAs(5
1Lg)
forPTH, calcitonin, actin,ratglyceraldehyde-3 phosphatedehydrogenase antisense vitamin Dreceptor (VDR)RNA(synthesized from pGEM zf (+)vectorusing T7polymerase), and pBR322 whichwereimmobilizedtoHybond filters (Amersham International) usingaslot blot apparatus.Hybridization was performed at 65°C for 72 h. The filters were washed three timesatroomtemperature in2x SSC,0.2% SDS for 5 min, then washed once at55°C for 15 min. The blotswereexposed
to CURIX-RP2film(AGFA Corp., Orangeburg, NY) at-70°Cwith
intensifying screens for 14 d.Inaddition, the filterswereexposedfor 24 hto aBioImagingPlate andquantified byaBio-imaging analyzer
BAS2000(FujiPhoto FilmCo., Tokyo Japan).
In situhybridization. Formaldehydefixedparaffinembedded thyro-parathyroid gland sections (8
Ism)
werecollected on gelatine-coatedglass slides, dewaxed, anddehydratedingradedethanol solutions. Sec-tionswerehybridized usingDNAcolor kits for nonradioactive in situ
hybridization (Amersham International) usingarandomprimed fluo-rescein labeled DNA probe for PTH. For detection the slides were
incubated with antifluorescein alkalinephosphatase conjugatefollowed by the addition of NBT (nitro blue tetrazolium)/BCIP
(5-bromo4-chloro-3indolyl phosphate)substrate.Theresultingcoloredprecipitate
was visualized by light microscopy. In addition in situhybridization
wasperformed usingan RNA color kit(AmershamInternational)with antisense and sense RNAprobes forPTH.
Serummeasurements. Serumcalciumandphosphateweremeasured in a autoanalyzer (Hoffmann-LaRoche & Co., Nutley, NJ). Serum 1,25(OH)2D3levels weremeasuredby aradioreceptor assay (Incstar Corp., Stillwater, OK). SerumiPTHlevels were measured with a rat
immunoradiomimetricassay (Nichols, SanClemente, CA). Statistical analysiswasperformedonthe Macintoshprogram Statview512+ (Aba-cusConcepts, Inc., Berkeley, CA), using Students' unpaired two-tailed
ttest.The results arepresentedasthe mean±SEM.
Results
Animalstudies. After 3 wk of the diets the serumphosphate
was lower in the rats fed 0.02%
phosphate
than those fed aHigh Non"" Low Figure 1. Effect of
di-1 2 3 4 5 6 7 8 9 10 11 etaryphosphateonrat PTH mRNA (rPTH).
_PYN
-~ Weanlingrats were fedthedifferent diets for 3 wkbefore surgery. Gel
US-
blot analysis of totalRNAfrom thyropara-thyroid tissue from rats hybridized with 32P random primed cDNA for ratPTH and 18S RNA. Lanes 1-3, high phosphate, high calcium (1.2% phosphate, 1.2% calcium); lanes 4-7, controls (0.3% phosphate, 0.6% calcium);lanes 8-11, low phosphate (0.02% phosphate, 0.6% calcium). Each lanerepresents totalRNAfrom thyroparathyroid tissue of a single rat.
normal(0.3%)orahigh phosphate (1.2%) (Table I), although there was also an increase in their serum calciums, and a large increase in their serum 1,25(OH)2D3 levels (Table I). A high serumphosphateleads tohypocalcemia,therefore the diet with
ahigh phosphate (1.2%) included a high calcium (1.2%) to prevent thehypocalcemia. Rats fed this diet had normal serum phosphateandcalciumlevels(Table I). Levels ofPTHmRNA were much lower in the rats with the low serum phosphate, as comparedtothenormalserumphosphate rats (Fig. 1). The rats fedahigh phosphate, highcalcium diet had an increase in their PTH mRNA(Fig. 1). The filters were hybridized for 18S RNA
toconfirmequal loading ofthegels. Similar results were found inthreerepeatexperimentsinwhich rats were fed the different dietsfor3 wk and they are shown quantitatively in Fig. 2. The PTH mRNA levels inratson alowphosphate diet were 25 ±8% ofthecontrolrats(P<0.01). Ahighphosphate diet led to an increase in PTH mRNA levels (P < 0.01), despite the normal
serumphosphate levels in this group. Theeffect oflow phos-phatetodecrease PTH mRNA levelswas also presentafter4, 10,and 14 dofthediet(not shown). SerumiPTHlevels at 14 d incontrol rats were22.1±3.7 pg/ml, andin rats after 14 d ofalowphosphate diettheywere4.7±0.8pg/ml (P < 0.05), whichwere similarto those ofparathyroidectomized ratsat 7 d(3.4±1.0pg/ml) and 14 d(3.3±0.2 pg/ml). Serumcalcium andphosphate levels in rats fed alowphosphate dietat 14 d were the sameasat21 d (TableI).
To visualize the effect of a low phosphate diet on PTH mRNA in situhybridizationstudieswereperformed. Thyropara-thyroid tissue froma ratfedanormaldiet and froma ratfed
alowphosphate dietwasfixedtothesameglassslidetoallow
150 Figure 2. Effect of
di-"IN*I- etaryphosphateon PTH
mRNA levels ofrat
thy-Z~
t100o_roparathyroid
glands.Weanlingratswerefed thedifferent diets for 3 wkbeforesurgery.The
50-°SO ,", mean ±SEfor three
dif-%
,_ ferentexperimentsis
shown, with threetofour
-Low Normal High rats ineachdietarygroup
-Low Normal High inthedifferent experi-Dietary Phosphate ments. ThePTH mRNA
levels are shown for ratsonlow, normal,andhighphosphatediets (asinFig. 1). *P
Figure3. Insituhybridizationof
parathyroid-thyroidsections with PTHprobe. (A) Parathyroid-thyroid tissue fromacontrolratwiththe
parathyroid (P) staining darklyfor thePTHmRNA. There isno
stain-ingof thethyroid (T) (x125). (B) Ahigher powerview of one A
showingtheparathyroidcells(P)
andthyroidfollicles(T) (X500). (C) Parathyroid-thyroidtissue froma ratwhich had been fedalow
phosphatediet for 2 wkshowingthe muchlighter stainingforPTH mRNA in theparathyroid (P)and nonepresentinthethyroid (T) (x125). (D)Ahigherpower view of C(x500).
comparison of the in situ signal. PIH mRNA was localized solelytotheparathyroidwith none present in thesurrounding thyroid(Fig. 3). Ahighpowerview showed thatPTHmRNA waslocalizedtotheperipheryofthe parathyroidcellsandwas present unformly in all parathyroid cells. In a rat whichhad been fed a low phosphate dietfor 2 wk there was much less PITH mRNA in the parathyroid. The PTH mRNA was still localizedto theperiphery oftheparathyroid cells andthe re-ducedmRNAaffectedallthecells.Identical resultswerefound in fourothersets of glands (notshown).In situhybridization usingan RNAantisense probe for PTH showedthe same result and therewas no labeling with the control sense RNA probe (notshown).
To separate theeffectof lowphosphatefrom that of a high serumcalcium, groupsofweanling rats werefedadiet which wasvitamin D-deficient,with a low content of bothphosphate (0.02%) and calcium (0.02%) for 10and 14 d(Fig.4). The serumphosphates weremarkedly reduced at 10 and 14 d and the serum calciums did notincreaseatboth 10 and 14 d (Fig. 4). There was anincrease inserum 1,25(OH)2D levels (Fig. 4).PTHmRNA and serumiPTHlevels weredecreasedtothe samedegreeasfor ratsfed the low phosphate diet with a normal calcium content.
Vitamin
D-deficient
rats. Second generation vitamin D-deficient rats fed a low phosphate diet for 10 and 21 d had increased levels of serum calcium,decreasedserumphosphates, and the same levels of serum 1,25(OH)2D3
(Table H). PTH mRNAlevels were markedly decreased in the vitamin D-defi-cientrats fed the lowphosphate diets for both 10 d (Fig. 5)and 3 wk(not shown) despitethe normal serum 1,25(OH)2D3 levels (Table II).
In a further experiment second generation vitamin D-defi-cientratswerefed diets with both a low calcium (0.02%) and phosphate(0.02%)or anormalcalcium (0.6%) andphosphate (0.3%). At 1 d there was alow serum phosphate with normal serumcalcium and 1,25(OH)2Dlevels in the rats fed the low calcium, low phosphate diet as compared to their controls fed anormalcalcium, normal phosphate diet (Fig. 6). PTH mRNA levels were markedly reduced (Fig. 6). There was nochange inacontrol gene 18S RNA(10.9±2.9: 11.8±0.8 OD units, P = NS). Therefore hypophosphatemia decreased PTH mRNA withnochangein serumcalciumor1,25(OH)2D.
Nuclear transcript run-ons. Nucleartranscriptrun-on assays wereperformed on nuclei from thethyroparathyroidtissue from groups of weanling rats fed normal and lowphosphate dietsfor 2 wk. Hybridization of the labeled RNAsynthesized in vitroto cDNAs immobilized on filters showed that there was no differ-encein the transcription of PTH from rats on a low phosphate diet as compared to a normal diet(Fig. 7).There was also no difference in the transcription of two control genes actin and ratglyceraldehyde-3 phosphatedehydrogenase, aswell as the 1,25(OH)2D3receptor(VDR)and calcitonin genes. These re-sults were confirmed quantitatively by an image analyzer. A repeatexperiment demonstrated identical results.
Discussion
TableII. The Effects of 10 and21d of Diets Given to Second Generation VitaminD-DeficientWeanling Rats
Serumphosphate Serum calcium Serum1,25(OH)2D3 (mean±SE[n]) (mean±SE[n]) (mean±SE[n])
mg/dl -Ca-Pi -Ca -Pi
10 days 14days Normal phosphate, normalcalcium for 10 d Low phosphate,
normal calcium for 10 d Normal phosphate,
normal calcium for 21 d Lowphosphate,
normal calcium for 21 d
mg/dl
12.0±3.0 (4) 10.5±0.3(4) 56.0±3.0(4)
4.5±0.5(4)* 17.2±0.7 (4)*
9.6±0.9(4) 10.5±0.4(4)
1.9±0.5(4)* 16.1±0.6(4)*
Control Diet Diet 10 days 10 days 14 days
IZ 20T
"TT
10
Control Diet Diet 10 days 10 days 14 days
Figure 4. Effect ofadietwithboth lowphosphateandcalciumon serumcalcium, phosphate, 1,25(OH)2D,andiPTHandPTHmRNA
levelsatdays 10 and 14.(a)Gel blotanalysisoftotal RNA from thyroparathyroidtissue fromratshybridizedwith 32P randomprimed cDNA forratPTH and 18S RNA.Lanes1-4, controlat10d; lanes 5-9,lowcalcium(0.02%)lowphosphate (0.02%)at10d;lanes
10-13,low calcium lowphosphate at14 d.(b)Bargraphoflevels of serumcalcium(0), phosphate (n),and 1,25(OH)2D (01)whichare
shownabove, andserumiPTH(0)and tissuePTHmRNA(a)below. The dataarerepresentedasday 10 forcontroldiet,and 10and14of theexperimentaldiet.Eachpointrepresentsthemean±SE offourrats.
WherenoSE is shown it is because the SEwassmaller thanthe line.
*P<0.001; **P< 0.05.
phosphate diet was dramatic with a marked decrease inPTH
mRNA levels and adecrease in serum iPTH levels to levels similar to those ofrats after parathyroidectomy. Rats on the
lowdietaryphosphatenotonly had lowserumphosphatelevels,
butalsohad increasedserumcalcium and 1,25 (OH)2D3 levels, inadditiontotheir decreased PTH mRNA levels. To separate
theeffect ofhypophosphatemiafrom that ofhypercalcemiaafter
alowphosphate diet, weanlingratswerefedadietdeficientin
vitaminDandwithalowphosphateand calciumcontent.These
rats developed hypophosphatemia with no increase in serum
n,number ofrats. *P< 0.01,or tNS comparedwithnormal phos-phate, normalcalcium for thesametimeperiod.
calcium and their serum iPTH and PTH mRNA levels were
reducedasbefore (Fig. 4). Howeverserum1,25 (OH)2D levels
intheseratswereincreased.
1,25 (OH)2D3 isanimportant factor in decreasing PTHgene
transcription both in vivo in rats and in vitro in parathyroid cells. It was, therefore, possible that hypophosphatemia
in-creased1,25 (OH)2D3 levels which then decreased PTH mRNA levels. To determine if the effect of phosphate was mediated
by 1,25 (OH)2D3 we performed studies on second generation
vitamin D-deficient rats.These ratsfed alow phosphate diet
from weaning had lowserumphosphates and highserum
calci-umsand nodifferences in theirserum 1,25 (OH)2D3 levels as
comparedtotheratsfedanormalphosphate in their diets (Table
II) and their PTH mRNA levels were still markedly reduced
(Fig. 5).Thisindicatesthat theeffect of phosphate in decreas-ing PTH gene expression, was not mediated solely by 1,25(OH)2D3,and the effect of1,25 (OH)2D3 would therefore be an additional effect. To determine whether
hypophos-phatemia alone withnohypercalcemiaandnoincrease inserum
1,25(OH)2D regulated the parathyroid gland, weanling rats
werefedadiet deficient in vitamin D and withalowphosphate and calciumcontent.After 1 d of this diet therewasadecrease
in serumphosphate and thyroparathyroid PTH mRNA levels
withnochange in serumcalciumor1,25 (OH)2D levels (Fig.
I
normal I lowPTH-1lS
-*Figure5. Effect of
di-etaryphosphateonPTH
mRNA levels in second generationvitamin D-deficientratsfed either normalorlowphosphate
dietsfor 10 d.18SRNA isshownasacontrol for theamountofRNAon
thefilter. Each lane
rep-resentstotal RNA from thyroparathyroidtissue ofasinglerat.
a
PTH
-NCa NPi IOdays
185 --a
b
IS
-I
lo-S
K
S-C
I I
15W
pg/ml
300 l
200 0
'100
"t
m
54.7±9.3
(4)*
58.2±4.0(4)
58.0±4.0(4)t
T -1s Figure
6.
Effectof di-T X etary phosphateonPTH::.:.:...YmRNAB>levels in second::.i m
6-
.1 _
| 10 generation vitaminD-deficientratsfed a
vita-4- s min D-deficient dietnu
.fiT.
with bothalowphos-S S ~~~~~~~~
~phate
and calciumcon-Pk 2- g * s g tentfor 1 d. Levels of
se-rumphosphateand PTH
0.C oo mRNA(top),andserum
Control Low Pi ControlLow Pi calciumand
10 100
1,25(OH)2D3
(bottom) T^*'
forratsfedavitaminD-75 S
9deficient,
n
my
%
W normalphos-phate,
normal calcium_1s
diet(controlgroup), ors so a vitamin
D-deficient,
X lowphosphate, low
cal-'
ciumdiet forld. There-sultsarethe mean±SE for fiveratsineach ControlLow Pi ControlLow Pi 0 group; *Pfor
1,25(OH)2D3
5
0.01. Datarepre-sentpooled serum fromtwo orthreeratswith three samples for controlratsandtwo samples for low phosphaterats.
6).This indicates that
hypophosphatemia
itselfdecreases PTH mRNAlevels withoutacontribution of calciumorvitaminD. We provided further evidence that the effect of hypophos-phatemiawas not mediatedbyvitamin Dby showingthatthe effect was not transcriptional. Theeffect of 1,25 (OH)2D3 on the PTH gene istranscriptional (2)andtherefore, ifthe effect ofphosphatewasmediatedby itseffectonserum1,25 (OH)2D3 levels, then it would be a transcriptional effect. We had pre-viously shown in vivo in rats injected with single doses of 1,25 (OH)2D3 that PTH transcription rates decreased to 10% of controls (2). We performed nuclear transcript run-ons on thyroparathyroid tissue fromrats fed a normal or phosphate-deficient diet which showed that the effect oflow phosphate wasnottranscriptional(Fig. 7). Thisresult furthersupportsthe conclusionthat theeffectof phosphateon PTHgeneexpression is notmediated by 1,25(OH)2D3.We performedin situ hybridization tovisualize the effect ofalow phosphate dietonPTHmRNA levels (Fig. 3). In a control rat PTH mRNA waspresent in all theparathyroidcells and localized to theperiphery of the cell. Thislocalization of mRNAswithin acell is oftenrelated to theirtrafficking within the cell (13) and the particular position of the PTH mRNA mightrepresent a stage initspassage onto translation and avail-ability for secretion. After a low phosphate diet there was a uniformand markedreductioninPTHmRNA(Fig.3).
Notonly did a low phosphate decrease PTH mRNA levels in vivo, but a high phosphate diet led to an increase in PTH mRNAlevels,despite the finding that the rats were not hyper-phosphatemic.Theeffect of thehighphosphate diet might pos-siblyreflect a transient
hyperphosphatemia
whichwas not de-tected in the present study, or a change in intracellular phosphate which wasnot studiedhere. Thepresentstudies therefore show thatphosphate regulatesPTH gene expression. Phosphate has been showntohave aneffect onserumPTHlevelsin anumber of studies.Lopez-Hilker
et al.have shown in dogs withexperi-low
normal
phosphate
PTH
CT
ACTIN
GPDN
pBR322
VDR
Figure7. Nuclear tran-scriptrun-ons for PTH, calcitonin, GPDN5, ac-tin, VDR,and pBR322 of rats fed normal andlow phosphate diets for 10d after weaning. The thyro-parathyroid tissue from
10 rats was pooled in each group, and nuclei isolated from each of the two preparations.
mentalchronicrenal failure thatphosphate restriction corrected their secondaryhyperparathyroidism independent of changesin serumcalciumand1,25 (OH)2D3levels(14). Theydidthisby placing theuremic dogs on dietsdeficientin bothcalciumand phosphate,whichled to lower levels ofserumphosphate and calcium, with no increase in the low levels of serum 1,25
(OH)2D3.
Despite this there was a 70% decreasein PTH levels. Lopez-Hilker's study onthe effect ofalowphosphate dieton serum PTH levelssuggested that phosphatehad an effect ontheparathyroid
cellbyamechanismindependent of its effect onserum 1,25(OH)2D3 and calcium levels (14). Sherwoodetal. injected phosphate to cows and showed that phosphate caused no directeffect on PTH secretion, but stimulated the parathyroid glands by lowering the serum calcium (15). In those studies theeffect of hypophosphatemia was not investi-gated,nor was the effect of a moreprolonged phosphate infu-sionstudied.
Phosphate retention has long been considered to be im-portant to thepathogenesis ofthesecondary hyperparathyroid-ism ofchronic renal failure, and the resultantdisabling renal osteodystrophy. In the '70s Slatopolsky and Bricker showed in dogs with experimental chronic renal failure that dietary phosphate restriction prevented their secondary hyperparathy-roidism (16). Clinical studies (17) have demonstrated that phosphate restrictioninpatientswithchronicrenalinsufficiency is effectiveinpreventingtheincrease inserum PTH levels ( 17-21). The mechanism of this effect was not clear, although at least part of it was considered to be due to changes in serum
1,25(OH)2D3
concentrations. In vitro (10, 22) and in vivo (17, 23) phosphate directly regulated the production of 1,25(OH)2D3. A raised serum phosphate decreases serum 1,25(OH)2D3
levelsand serumcalcium byformation of calcium phosphate in the serum which is then deposited in bone and soft tissues.Therefore,
phosphate undoubtedly plays a central role in thepathogenesis of secondary hyperparathyroidism,both by its effecton serum 1,25(OH)2D3,
calciumlevels, and inde-pendently.Theparathyroids hyperfunctioninsecondary hyperparathy-roidism at a number of levels. Firstly, there is an increase in PTHsecretion, which is due to the low calcium of chronic renal failure. Itis also controversial whether there isanalteration in parathyroid cell sensitivity (set point) to levels of calcium which wouldnormally decreasePTHsecretion (24, 25). PTH synthesis is increased because of increasedPTH gene expres-sion per cell (8), and also in the long term, because of an
Regulatlon
of PTH Gene Expresslonlow phosphorus low calcium
PTH mRNA
1
,25(OH)2VitaminD
estrogensFigure 8.The regulation of PTH gene expression. Hypocalcemia and estrogensincrease PTHmRNAlevels. 1,25(OH)2D3and hypophos-phatemiadecrease PTH mRNA levels. Not shown is the effect of a high phosphate to increase PTH mRNA levels.
showed thatweanlingratsmaintained for 3wk ondietsdeficient invitaminDand calciumhad a10-fold increasein PTH mRNA levels, whichwasmainly dueto anincrease inPTH gene expres-sionpercell withamuch smallercontribution of increased cell number(8). They,therefore, demonstratedthatchanges inPTH geneexpression are animportant factorin the pathogenesis of secondaryhyperparathyroidism.The presentstudy indicates that changesin serumphosphate alsoregulatePTH geneexpression eitherbyadirectorindirect actionon theparathyroid cell,and araised serumphosphate would increase PTH mRNA levels and contributetothepathogenesis ofsecondary hyperparathy-roidism.
It is not known how phosphate would directly affectthe parathyroid cell. Phosphate equilibrates acrosscell membranes
by
active and inactive transportmechanisms,
andthe changes in extracellular phosphate might very well be reflected by changes in intracellular phosphate, as well asby
changes in membranephospholipid
composition. Forexample,
one phos-pholipid, inositoltriphosphate isanimportantsecond messenger in all cells(26), including the parathyroid(27, 28).
Bourdeau et al. have shown thatboth
calcium and1,25
(OH)2D3
ledtorapid (5 s) increases inphosphatidinyl metabolites in
porcine
parathyroid cells(29).Itwould beintriguing todetermine the interactionsintheparathyroidcellof changesin
calcium,
phos-phate, and1,25(OH)2D3.
Theeffect of
changes
inserumcalciumonPTH gene expres-sion and secretionarebetter understood. We(6, 8)
and Yama-moto et al.(7) haveshown thatalow serumcalcium leadstomarked increases in PTH mRNA levels.
However,
we have increased serumcalcium byanumber of methods forperiods
from 6h to 3 wk and showed thata
high
serumcalcium had no effect onPTHmRNA levelsinvivo, despiteserumcalciums as high as 25 mg/dl (6, 30) although a modest effect of acalcium infusion for 48 h has been demonstrated(7).The mech-anism of calcium sensing by theparathyroid cell isthrough a
calcium receptor which isG-proteincoupledandactivates inosi-toltriphosphate production (31).Studies on the action of phos-phate on theparathyroid cell in vitro, for instance in primary cultures ofbovine
parathryoid
cells,have notproduced positive results whichwould allow studies on asignaltransduction path-way. It is possiblethat the effectofphosphate is directly onthe parathyroid cell or it isjust as reasonable tohypothesize that it is anindirect effectbyamechanismnotinvolvingcalcium
orvitamin D.
Changes
inextracellularphosphate
concentration dodirectly
produce
changes
in cells.Forinstance,
alowphos-phate mediumleads to an increase in phosphate transport by renal andother cells in culture (32-34) which is not secondary tochanges in ATP or ADP concentration (35, 36), and is associ-ated with a decrease inintracellularcalcium concentration (37). The physiology of phosphate effect on the parathyroid remains tobe elucidated.
In anyevent it is now clear that the parathyroid cell, and particularly the expression of the parathyroid gene respond to
anumber offactors,such as 1,25 (OH )2D3, calcium, estrogens, and now phosphate (Fig. 8). Changes in the concentrations of these factors lead to the altered synthesis and secretion of PTH, which is important to the pathogenesis of disabling diseases such as renal osteodystrophy and osteoporosis. The effective prevention and treatment of these diseases is dependent on ad-vances in ourunderstanding of theirpathophysiology, such as how the expression of the PTH gene is regulated by phosphate.
Acknowledgments
The authors acknowledge Ms. MiriamOffner for excellent technical assistance, S. Edelstein ofthe WeizmannInstitue for performing the serum 1,25(OH)2D3 assaysandto C. R. Kleeman forhelpful discus-sions.
This workwassupportedbytheThyssenFoundation.
References
1.Silver, J. 1992. Regulation of parathyroid hormone synthesis and secretion. InDisorders of Bone and MineralMetabolism,F.L. Coe and M. J. Favus, editors Raven Press, Ltd., New York. 83-106.
2.Silver, J., T.Naveh-Many,H. Mayer, H. J. Schmelzer and M. M. Popovtzer. 1986. Regulationbyvitamin D metabolites ofparathyroidhormonegene transcrip-tion in vivo in therat.J. Clin. Invest.78:1296-1301.
3.Silver, J., J.Russell, and L. M. Sherwood. 1985. Regulation by vitamin D metabolites ofmessengerribonucleic acid for preproparathyroidhormonein iso-lated bovineparathyroidcells.Proc. Natl. Acad. Sci. USA.82:4270-4273.
4.Okazaki, T.,T.Igarashi, and H. M.Kronenberg.1988. 5'-flankingregion of the parathyroid hormonegenemediates negative regulationby 1,25 (OH)2 vitamin D3. J. Biol.Chem.263:2203-2208.
5. Demay, M. B., M. S. Kiernan,H. F.DeLuca, and H. M. Kronenberg. 1992. Sequences in the human parathyroid hormone genethat bind the
1,25-dihydroxyvitaminD3receptorand mediatetranscriptionalrepression inresponse
to1,25-dihydroxyvitaminD3.Proc. Natl. Acad. Sci. USA. 89:8097-8101. 6.Naveh-Many,T., M. M.Friedlaender,H.Mayer,and J. Silver. 1989. Cal-cium regulates parathyroid hormonemessengerribonucleic acid (mRNA), but
notcalcitonin mRNA in vivo in therat.Dominant role of1,25-dihydroxyvitamin
D.Endocrinology. 125:275-280.
7.Yamamoto,M.,T.Igarashi, M.Muramatsu,M.Fukagawa,T.Motokura, and E. Ogata. 1989. Hypocalcemia increases and hypercalcemia decreases the steady-state level of parathyroid hormone messengerRNA intherat.J. Clin. Invest. 83:1053-1056.
8.Naveh-Many,T.,and J.Silver. 1990.Regulation ofparathyroidhormone geneexpressionbyhypocalcemia, hypercalcemia,and vitamin Dintherat. J. Clin. Invest. 86:1313-1319.
9.Naveh-Many,T., G.Almogi,N.Livni, and J. Silver. 1992.Estrogen
recep-torsandbiologicresponseinratparathyroid tissueand C-cells. J. Clin. Invest. 90:2434-2438.
10.Tanaka, Y., and H. F. DeLuca. 1973. The control of vitamin D by inorganic
phosphorus.Arch. Biochem.Biophys.154:566-570.
11. Raynolds, M. V.,P. D. Awald,D. F.Gordon, A.Guierrez-Hartmann,
D. C. Rule, W. M.Wood,and R. H. Eckel.1990.Lipoproteinlipasegene
expres-sioninratadipocytesisregulated by isoproterenoland insulinthroughdifferent
mechanisms. Mol. Endocrinol. 4:1416-1422.
12.Groudine,M.,M.Peretz,and H. Weintraub. 1981.Transcriptional regula-tion of hemoglobin switchinginchicken embryos.Mol. Cell.Biol. 1:281-288.
13. Lawrence,J. B., and R. H. Singer. 1986. Intracellular localization of messengerRNAs for cytoskeletal proteins.Cell. 45:407-415.
14.Lopez-Hilker,S.,A. S.Dusso,N. S.Rapp,K. J.Martin,and E.Slatopolsky. 1990. Phosphorus restrictionreverseshyperparathyroidismin uremiaindependent of changesin calcium and calcitriol. Am. J.Physiol.259:F432-F437.
15. Sherwood,L. M.,G. P.Ayer,C. F. Ramberg, D. S.Kronfeld,G. D.
proportional control by calcium, lack of effect of phosphate. Endocrinology. 83:1043-1051.
16.Slatopolsky, E., and N. S. Bricker. 1973. The role of phosphorus restriction in theprevention of secondary hyperparathyroidism in chronic renal disease. KidneyInt. 4:141-145.
17.Portale, A. A., B. E. Booth, B.P.Halloran, and R. C. J. Morris. 1984. Effect of dietary phosphorus on circulating concentrations of 1,25-dihydroxyvitamin D andimmunoreactive parathyroid hormone in children with moderate renal insufficiency. J. Clin. Invest. 73:1580-1589.
18.Lucas, P.A., R. C. Brown, J. S.Woudhead, and G. A.Coles. 1986. 1,25-dihydroxycholecalciferol and parathyroid hormoneinadvanced chronic renal
failure:effects of simultaneousproteinandphosphorus restriction. Clin. Nephrol. 25:7-10.
19. Lafage, M. H., C. Combe, A. Fournier, andM.Aparicio. 1992. Ketodiet, physiological calcium intake and native vitaminDimprove renal osteodystrophy. KidneyInt. 42:1217-1225.
20. Combe, C., and M. Aparicio. 1994. Phosphorus and protein restriction andparathyroid function in chronic renal failure. Kidney Int. 46:1381-1386.
21.Aparicio,M., C. Combe, M. H. Lafage,V. DePrecigout, L. Potaux,and
J. L.Bouchet. 1994. In advanced renal failure, dietary phosphorus restriction reverses hyperparathyroidism independent of the levels of calcitriol. Nephron. 63:122-123.
22.Condamine, L., F. Vztovsnik, G. Friedlander, C. Menaa, andM. Garabe-dian. 1994. Local action of phosphate depletion and insulin-like growth factor 1 on invitroproduction of 1,25-dihydroxyvitaminDby culturedmammaliankidney cells. J. Clin. Invest. 94:1673-1679.
23. Portale, A. A., B. P. Halloran, and J. Curtis Morris. 1989. Physiologic regulation of the serum concentration of 1,25-dihydroxyvitamin D by phosphorus innormalmen. J. Clin. Invest.83:1494-1499.
24. Brown,E.M.,R. E.Wilson,R.C.Eastman, J. Pallotta, and S.P.Marynick. 1982. Abnormal regulation of parathyroidhormonereleaseby calcium in second-aryhyperparathyroidism due to chronicrenal failure. J. Clin. Endocrinol. & Metab. 54:172-179.
25.Ramirez, J. A.,W.G. Goodman,J.Gornbein,C.Menezes,L.Moulton, G.V. Segre,and L.B.Salusky. 1993.Direct in vivocomparison of calcium-regulated parathyroid hormone secretioninnormal volunteers andpatients with secondaryhyperparathyroidism. J. Clin. Endocrinol. & Metab. 76:1489-1494.
26.Berridge, M. J., and R. F. Irvine. 1989. Inositol phosphates and cell signalling. Nature(LondL).341:197-206.
27. Brown, E. M., J. Redgrave, and J. Thatcher. 1984. Effect of the phorbol ester TPA onPTH secretion. Evidence for a role for protein kinase C in the control ofPTH release. FEBS (Fed Eur. Biochem Soc.)Lett. 175:72-75.
28. Brown, E. M. 1991. Extracellular Ca2+ sensing, regulation of parathyroid cellfunction, and role of Ca2+ and other ionsasextracellular(first) messengers. Physiol. Rev.71:371-411.
29. Bourdeau, A., J.-C. Souberbielle, P. Bonnet, P. Herviaux, C. Sachs, and M. Lieberherr. 1992. Phospholipase-A2 action and arachidonic acid in calcium-mediatedparathyroid hormone secretion. Endocrinology. 130:1339-1344.
30. Naveh-Many, T., F. Raue,A.Grauer, and J. Silver. 1992. Regulation of calcitoningene expression byhypocalcemia,hypercalcemia, and vitaminDin the
rat. J.Bone.Miner. Res.7:1233-1237.
31.Brown,E.M.,G. Gamba,R.Riccardi,M.Lombardi,R.Butters, 0. Kifor, A.Sun, M. A. Hediger, J. Lytton, and J. Hebert. 1993. Cloningand characteriza-tion of an extracellularCa2+-sensingreceptor from bovineparathyroid.Nature
(Lond). 366:575-580.
32.Caverzasio, J., C. D. A. Brown, J. Biber, and J.-P. Bonjour. 1985. Adapta-tionof phosphate transport in phosphate-deprived LLC-PK, cells.AmLJ.Physiol. 248:F122-F127.
33. Biber, J., J. Forgo,and H. Murer.1988.Modulation of Na+-Pi cotransport in opossum kidney cells by extracellular phosphate. AnL J. Physiol. 255:C155-C161.
34. Escoubet, B., K. Djabali, and C. Amiel. 1989. AdaptationtoPideprivation
of cellNa-dependent P, uptake:awidespread process. AnL J. Physiol. 256:C322-C328.
35. Barac-Nieto, M., T. L. Dowd, R. K. Gupta, and A. Spitzer. 1991. Changes inNMR-visible kidney cell phosphate with age and diet: relationship to phosphate transport.Am.J.Physiol.261:F153-F162.
36.Barac-Nieto, M., H.Corey, S. M. Liu, and A.Spitzer. 1993. Role of intracellular phosphate in the regulation of renal phosphate transport during devel-opment. Pediatr. Nephrol. 7:819-822.
37. Escoubet, B., M. C. Garestier,C.Le-Grimellec, and C. Amiel. 1993.
Multiple modulationofNa-dependent Pi uptake by cellular Ca in MDCK cells.
