American Journal ofPathology, Vol. 150, No. 1, January 1997 Copyright © American Societyfor Investigative Pathology Short Communication Prolactin-Derived Amyloid in the Aging Pituitary Gland

Per Westermark, Lars Eriksson, lated and their nature determined by amino acid Ulla Engstrom, Sverker Enestr6m, and sequence analysis. Most of the characterized local- Knut Sletten ized amyloids have turned out to be derived from From the Department ofPathology, Linkoping University, polypeptide hormones.7 To date, four different hor- Linkoping, the Department ofPathology, Uppsala University, mones are known to give rise to amyloid in humans, Uppsala, and the Ludwig Institute of Cancer Research, namely, calcitonin in medullary carcinoma of the thy- Uppsala Branch, Uppsala, Sweden; and the Department of roid,6 islet amyloid polypeptide (IAPP) in insulinomas Biochemistry/Biotechnology Center of Oslo, University of and islets of Langerhans,9 12 atrial natriuretic factor Oslo, Oslo, Norway (ANF) in the atria,13,14 and rarely, insulin subcutane- ously at injection sites.15 These four hormones are not closely related, and the reason they aggregate into amyloid fibrils is still obscure. However, there is SmaU amyloid deposits occur commonly in differ- growing evidence that inherent amyloidogenic se- ent organs in association with aging. As in other quences in the fibril proteins are a prerequisite of amyloids, the in the age-associatedforms fibrils amyloid formation, but additional factors, yet to be are built up by proteins, unique to every specijc defined, are obviously also important. histological type. The amyloidproteins that have As amyloid commonly occurs in some other hor- been in localized amyloid of human identified mone-producing organs, more amyloid-forming endocrine organs have aU been of polypeptide polypeptide hormones are expected to be eluci- hormone nature, and these include calcitonin, dated. In this paper, we present results of the very islet amyloid polypeptide (amylin), and atrial prevalent amyloid form in the anterior lobe of pituitary natriuretic factor. In the present study, we add glands of aging individuals. prolactin to the increasing group ofknown amy- loid proteins and show that this hormone con- stitutes the amyloid ftbrils of pituitary glands of aging individuals. (Am J Pathol 1997, Materials and Methods 150:67-73) Material Whole pituitary glands were removed at autopsies Small deposits of amyloid constitute a typical age- from patients without systemic amyloidosis. The pi- related phenomenon in many different organs and tuitaries were divided and a sagittal section was tissues. Well known examples are the cerebral amy- fixed in 10% buffered neutral formalin and embed- loid seen in normal aging and in Alzheimer demen- ded in paraffin. The rest of the glands were frozen at tia1-4 and the amyloid in the islets of Langerhans, -20°C and stored until used for extraction. In some found mainly in non-insulin-dependent diabetic indi- instances, small pieces were fixed in 2% paraformal- viduals but also in nondiabetic persons.5'6 There is increasing evidence that the localized amyloid de- Supported by the Swedish Medical Research Council (project 5941) posits found in many different organs in association and the Research Council of Norway. with aging are chemically very diverse. Although Accepted for publication August 27, 1996. most of these amyloid forms are not yet character- Address reprint requests to Dr. Per Westermark, Department of ized, several amyloid fibril proteins have been iso- Pathology I, University Hospital, S-581 85 LinkOping, Sweden.

67 68 Westermark et al AJPJanuary 1997, Vol. 150, No. 1

100 VO0 A I IlI 30 50 70 4~~ - 'Cui550 Fraction number Figure 1. Gelfiltration ofpituitary amyloidflbrils. No retardedprotein peak occurred. The bar indicates pooledfractions in which an amy- loid-specific protein was identified by RP-HPLC. VO void volume; Ar- row: total volume. Absorbance is in arbitrary units.

0 30 dehyde and 1% glutaraldehyde, pH 7.2, and em- Time (minutes) bedded in Lowicryl K4M (Polaron, Waldkraiborg, Figure 2. RP-HPLCperformed ongelfiltrationfractions as indicated in Germany) for immune electron microscopic investi- Figure 1. Thepeak indicated by A was uniquefor the amyloid mate- rial, whereas the other peaks were seen also when an amyloid-free gation. material was run on the column. Absorbance is in arbitrary units. One amyloid-rich pituitary (male, 86 years) and one withou4 amyloid deposits (female, 81 years) were homogenized individually and repeatedly in compared after comparable fractions from the two 0.15 mol/L sodium chloride followed by distilled wa- gel filtrations had been run. In this way, a single peak ter as described.11 However, for a better solubiliza- was identified that was seen with the amyloid-rich tion of secretory granules that could contaminate the material but not with the control pituitary material final fibril preparation, an extra homogenization step (Figure 2). This peak material was purified on the with 10% acetic acid was inserted before the last same column by repeated injections (20 x 200 ,ul) of water homogenization. The pellets, containing the the rest of the gel filtration material. It was collected, amyloid (verified by Congo red staining of a droplet dried, and used for amino acid sequence analysis. dried on a glass slide), were lyophilized and utilized for other studies. Amino Acid Sequence Analysis Amyloid Protein Identification and Amino-terminal amino acid sequence analysis was Purification performed with the aid of an automatic protein se- quencer (477 A, Applied Biosystems, Foster City, The lyophilized material (approximately 10 mg in CA) coupled to a phenylthiohydantoin amino acid each case) was dissolved overnight in 6 mol/L gua- analyzer. nidine/HCI containing 0.1 mol/L Tris/HCI, pH 8.0, 0.02 mol/L EDTA, and 0.1 mol/L dithiothreitol. After Synthetic Peptides centrifugation, the supernatant was gel filtered in 5 mol/L guanidine/HCI in distilled water through a Three different 15-residue-long, carboxyl-terminally 1.0 x 120 cm Sepharose 6B (Pharmacia, Uppsala, amidated peptides, corresponding to amino resi- Sweden) column at a flow rate of 2 ml/hour and dues 7 to 21, 20 to 34, and 43 to 57 of human monitored at 280 nm. Fractions of 1 ml were col- prolactin, were synthesized as described.16 Analysis lected. by mass spectrometry revealed the expected molec- As no peaks, except for the void volume peaks, ular masses. were seen for any of the materials (Figure 1), map- ping by reversed-phase high performance liquid Formation of Fibrils in Vitro chromatography (RP-HPLC) was performed. For this, pooled fractions starting close to fractions at The capacity of the three synthetic peptides to form total column volume were directly injected in 200-,ul fibrils in vitro was tested as described.16'17 In brief, samples onto a Brownlee Aquapore 30 x 4.6 mm C4 the peptides were dissolved at 10 mg/ml in 10% column and then eluted with a linear 0 to 100% acetic acid and incubated overnight at room temper- gradient of 70% acetonitrile in 0.1% trifluoroacetic ature followed by neutralization with a concentrated acid at a flow rate of 0.4 ml/minutes. The elution was solution of ammonia. Formation of amyloid-like fibril- monitored at 226 nm, and the elution patterns were lar material was assessed by examination in polar- Prolactin Amyloid 69 AJPJanuary 1997, Vol. 150, No. 1 ized light of Congo-red-stained dried droplets and Results by examination of material in a Jeol electron micro- scope at 80 kV. For the latter, material was applied to Amyloid Deposits formvar-coated copper grids and negatively con- The localized amyloid deposits identified in the pitu- trasted with sodium phosphotungstate. itary glands were usually scarce and had a soft, noncompact appearance. They stained weakly with Congo red and showed a faint but clear green bire- Immunohistochemistry and Immune fringence. Electron microscopically, amyloid depos- Electron Microscopy its were identified as extracellular, fairly short, and rigid fibrils without any obvious orientation. The di- The synthetic peptides were coupled to keyhole lim- ameter of the fibrils varied between 10 and 20 nm. A pet hemocyanin (Sigma Chemical Co., St. Louis, pituitary gland with a typical but comparatively large MO), using 1-ethyl-3-(3-dimethylaminopropyl)-car- amount of amyloid was chosen for amyloid purifica- bodiimide (Sigma) as coupling agent. Antisera were tion. raised in three rabbits by immunization with the cou- pled peptides, and the titers of the antisera were checked by enzyme-linked immunosorbent assay, Identification and Amino Acid Sequence using the uncoupled peptide as antigen. Antiserum Analysis of the Amyloid Fibril Protein to human prolactin was from Dako (Glostrup, Den- mark). Initial experiments, in which SDS-PAGE results of For immunohistochemistry, deparaffinized sec- amyloid-rich pituitary extracts were compared with failed tions of two pituitaries, different from those used for extracts from pituitary glands without amyloid protein purification and containing large deposits of to identify a specific amyloid protein band (unpub- amyloid, were incubated with the primary antisera, lished results). Likewise, gel filtration of the two pitu- diluted 1:100 to 1:400. Some sections were pre- itary extracts studied in the present investigation treated for 10 minutes by microwave (640 W) in showed no retarded protein peak in any of the ma- 0.01% sodium citrate buffer, pH 6.0, before applica- terials (Figure 1). Therefore, fractions from the gel tion of the antiserum. The reaction was visualized by filtrations were analyzed systematically by RP-HPLC the biotin-avidin method. For controls, the primary and comparisons of the elution patterns were made. antiserum was replaced with preimmune serum or Using this strategy, a distinct peak appeared when the primary antiserum was absorbed with the uncou- pooled fractions (approximate molecular mass pled antigen before the incubation. range, 10 to 25 kd) from the gel filtration of the Immune electron microscopy was performed on amyloid-rich material were run, and only this peak ultrathin Lowicryl sections and mounted on formvar- was lacking for the amyloid-free material (Figure 2). coated nickel grids as described.18 The primary an- Amino-terminal amino acid sequence analysis of this tiserum was diluted 1:100, and the reaction was peak material gave a sequence corresponding to visualized with protein A-gold (Auroprobe, 15-nm prolactin positions 1 to 32 (Figure 3). particles, Janssen, Beerse, Belgium). For control, antiserum preabsorbed with the uncoupled antigen Reaction ofAntisera with Pituitary Gland was used. Amyloid The commercial antiserum to prolactin did not react with the pituitary amyloid in immunohistochemistry or Electrophoretic Methods in immune electron microscopy. Therefore, rabbits Sodium dodecyl sulfate polyacrylamide gel electro- were immunized with synthetic peptides corre- phoresis (SDS-PAGE) was performed as de- sponding to segments of prolactin. All three syn- scribed.19 For immunoblotting, proteins were elec- thetic peptides gave rise to specific antisera as re- troblotted onto a nitrocellulose membrane (BioRad vealed by enzyme-linked immunosorbent assay. Laboratories, Richmond, CA) and incubated over- However, only the antiserum against prolactin(43-57) night with the primary antiserum diluted 1:200. reacted with amyloid when tested immunohisto- Bound antibodies were visualized with protein A cou- chemically, and only after microwave treatment (not pled to 20-nm gold particles followed by silver en- shown). The labeling was abolished by absorption hancement according to the manufacturer's descrip- with prolactin(43-57), and preimmune serum did not tion (BioCell, Cardiff, UK). react. 70 Westermark et al AJPJanuary 1997, Vol. 150, No. 1

10 20 30 A. LPI(C)PGGAAR(C)QVTLRDLFDRAVVLSHYIHNL

10 20 30 40 50 60 B. LPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLA

C. GAARCQVTLRDLFDR DRAVVLSHYIHNLSS RYTHGRGFITKAINS Figure 3. A: Amino-terminal amino acid sequence of an amyloidfibril protein, purified from an amyloid-rich pituitary gland. The sequence is identical to that ofprolactin, positions 1 to 32. The cysteine residues were notfully verifted. B: Tbe 64-amino-acid residue amino-terminal part of human prolactin. C: Amino acid sequences ofthe three synthetic peptides used in this study. Ofantisera made against the threepeptides, only that against prolactin( 43-57) reacted with amyloid deposits in immunohistochemical and immune electron microscopic studies.

Immune electron microscopically, antiserum Fibrils in Vitro against prolactin(43-57) labeled amyloid fibrils spe- cifically (Figure 4), and this was abolished by preab- At the tested concentration, all three synthetic pep- sorption with the synthetic peptide. A strong labeling tides were easily soluble in 10% acetic acid, and no did also occur on secretory granules of a few cells. gel particles were visible after incubation overnight. However, after neutralization with ammonia and overnight incubation, fibrils were found in all three SDS-PAGE and Immunoblotting preparations. In all cases, the fibrillar material had In SDS-PAGE, no amyloid-specific band was de- strong affinity for Congo red and showed green bi- tected on gels stained with Coomassie blue (not refringence. Electron microscopy showed more or shown). Immunoblotting analyses were performed less wavy fine fibrils, 4 to 5 nm in diameter, in all with amyloid fibril material of one pituitary with amy- three materials. loid and one sample of control material, using the antisera against prolactin(20-34) and prolactin(43- 57). With anti-prolactin(43-57), a band correspond- ing to the Mr of prolactin was seen in the amyloid- containing material, which was lacking for the control material (Figure 5). When anti-prolactin(20-34) was used, no reaction was obtained with any of the ma- terials (not shown).

Figure 5. Immunoblot analysis of pituitary amyloid fibril material (right lane) and materialfrom apituitary uithout amyloid (left lane). Figure 4. Electron microscopy ofa section ofthepituitary used alsofor A band corresponding to the expectedposition ofhumanprolactin (23 protein purification. 7be section was incubated with an antiserum kd) is evidentfor the amyloid-containing material only (arrow). Bars againstprolactin(43-57) and the reaction was visualized wtith protein correspond to molecular weight markers that are, from above, bovine A-goldparticles. The labeling ofamyloidfibrils is specific. Magnifica- serum albumin (67kd), ovalbumin (46 kd), soybean trepsin inhibitor tion, x 40, 000. (20.4 kd), and lysozyme (14.4 kd). Prolactin Amyloid 71 AJPJanuary 1997, Vol. 150, No. 1

Discussion mature full-length calcitonin32 and a fragment of pro- calcitonin have been found to be predominant pro- Among the age-related amyloid deposits, those oc- teins.8 ANF,18 IAPP,17 insulin,33'34 and calcitonin35 curring in polypeptide-hormone-producing tissues have all been shown to form amyloid-like fibrils in constitute a distinctive group.7 The polypeptide hor- vitro, indicating that the mature full-length protein mone-derived amyloid forms are very common in can make amyloid fibrils. It should be noted that all of some organs. Thus, some degree of islet amyloid- the synthetic peptides of prolactin that we tested osis is seen in at least 50% of elderly individuals and formed fibrils in vitro. However, these latter results in as much as 95% of persons with non-insulin-de- should be interpreted with care as the properties of pendent diabetes mellitus.20'21 Isolated atrial amy- small segments of a protein can differ compared with loid derived from ANF is found in 65% of persons the full-length protein. over 70 years of age.22 Amyloid deposits in the ad- A hypersecretion of the hormone is generally im- enohypophysis are very prevalent in aging individu- plicated in the pathogenesis of the polypeptide-hor- als, and the prevalence increases to 80 to 95% in mone-derived amyloids.7 The pituitary gland can persons over 80 years of age.23'24 Amyloid deposits maintain a prolactin secretory capacity at old age,36 are also commonly found in pituitary adenomas.2530 and age per se does not seem to alter the rate of In analogy with amyloid in the islets of Langerhans, secretion of prolactin in humans,37 nor is the circa- which is identical to amyloid commonly found in dian rhythms of prolactin plasma levels modified at insulinomas and consisting of IAPP,9'10 it is highly old age.38 A tendency of higher plasma levels of the probable that the chemical nature of pituitary gland hormone in men after 65 years was found by some amyloid and amyloid in pituitary adenomas are iden- authors.39,40 The same result was reported by tical.31 Although a hormonal nature of the pituitary Weiske and Frick,41 who measured the serum pro- adenoma amyloid has been suspected, immunohis- lactin concentration in different age groups and tochemical analyses with antibodies to pituitary hor- showed a small increase for men between 50 and 80 mones have been negative24'31 or given highly vari- years. The basal plasma levels of prolactin are, how- able results. Likewise, in the present study, no ever, generally found to be higher in women than in reaction with the amyloid was obtained with the com- men of the same age groups. It should be noted that mercial antiserum to prolactin. Only one antiserum to there is no significant sex difference in the preva- a synthetic peptide corresponding to a small part of lence of localized pituitary amyloidosis.24 prolactin immunoreacted. The reason for this is not Amyloid occurs in pituitary adenomas with varying clear, but it is possible that important epitopes are hormonal profiles. It seems to be most common in hidden within the amyloid fibril or have disappeared growth-hormone- and prolactin-secreting tu- due to conformational changes. Formation of amy- mors25 26'30 but is found also in adenomas of other loid fibrils sometimes includes a conformational shift types.30'42 Although prolactin is probable as the or- from a-helix to ,B-sheet. igin for most pituitary adenoma amyloid deposits, it Due to the small amyloid deposits in a small or- cannot be ruled out that other pituitary hormones gan, a direct analysis of localized pituitary amyloid may also have amyloidogenic potential. has been difficult. Only by systematic RP-HPLC pep- tide mapping of fractions obtained from gel filtration of the amyloid-containing material and comparison Acknowledgments with a corresponding amyloid-free pituitary material was it possible to identify the amyloid-specific pro- We thank Marie-Louise Eskilsson, Jessie Juul, and tein that was identified as prolactin by amino-termi- Helen Wilhelmsson for skilled assistance. nal amino acid sequence analysis. Many amyloid fibril proteins are fragments of larger protein precur- sors. Due to lack of material, no further characteriza- References tion of the amyloid prolactin could be performed by amino acid sequence analysis, but the immunoblot- 1. Glenner GG, Murphy MA: Amyloidosis of the nervous ting results indicate intact prolactin molecules rather system. J Neurol Sci 1989, 94:1-28 than 2. Stam FC, Wigboldus JM, Smeulders AWM: Age inci- fragments are present in the amyloid fibrils. This dence of senile brain amyloidosis. Pathol Res Pract is in accordance with most other polypeptide hor- 1986, 181:558-562 mone-derived amyloid proteins as ANF, IAPP, and 3. Mandybur TI: The incidence of cerebral amyloid angi- insulin extracted from amyloid have all been full- opathy in Alzheimer's disease. Neurology 1975, 25: length proteins. In calcitonin-derived amyloid, both 120-126 72 Westermark et al AJPJanuaty 1997, Vol. 150, No. 1

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