K562 Erythroid and HL60 Macrophage Differentiation Downregulates Polycystin, a Large Membrane-Associated Protein Gianluca Aguiari, Roberta Piva, Elisa Manzati, Elisa Mazzoni, Giuseppina Augello, Elisabetta Chiari,* Sabrina Moretti,† Luca Maria Neri,‡ and Laura del Senno 1 Dipartimento di Biochimica e Biologia Molecolare & Centro Interdipartimentale di Biotecnologie; ‡Sezione di Anatomia Umana, Dipartimento di Morfologia ed Embriologia; †Sezione di Ematologia, Dipartimento di Scienze Biomediche e Terapie Avanzate; and *Sezione di Genetica Umana, Dipartimento di Medicina Sperimentale e Diagnostica, Universita ´ degli Studi, Ferrara, Italy Polycystin, the PKD1 gene product mutated in autoso- mal dominant polycystic kidney disease, is a large mem- brane protein which is important in the differentiation of epithelial tubular structure. Furthermore, PKD1 mRNA is expressed in various tissues and in neoplastic cell lines particularly, suggesting that polycystin might be involved in differentiation and/or proliferation of other cell types. Therefore, in order to investigate such a possible role, polyclonal antibodies against a recombi- nant polycystin peptide were raised and used to study polycystin expression in human leukemia cell lines com- mitted to differentiation. Using Western blot and laser scanning confocal microscopy analyses, we demon- strated expression of polycystin in erythroleukemia K562 cells as a membrane-associated polypeptide of ap- proximately 450 kDa, mainly localized in cell-cell con- tacts. Protein size and subcellular distribution were sim- ilar to those found in the kidney epithelial KJ29 cell line. In addition, K562 cell erythroid differentiation induced by hemin was characterized by a reduction in polycystin expression, as measured by Western blot and Northern blot analyses. Cytofluorimetric analysis indicated that upon hemin treatment there was a progressive reduc- tion in the number of polycystin-expressing cells as well as in proliferation rate. Furthermore, reduction in pro- liferating and polycystin-expressing cells was also ob- served in K562 cells after serum starvation. When serum was added to the serum-deprived cells an increase in cell number as well as in number of polycystin-positive cells was observed. In addition, polycystin, also expressed in promyelocytic leukemia HL60 cells, was downregulated when macrophage differentiation in HL60 was induced by TPA. Therefore, in these leukemic cells downregula- tion of polycystin appeared to be closely related to re- duction in cell proliferation and to induction of differ- entiation. This suggests that polycystin may play a relevant role in these cell processes. © 1998 Academic Press Key Words: polycystin expression; myeloid cells; cell proliferation; differentiation. INTRODUCTION Polycystin, the product of the PKD1 gene mutated in 85% of autosomal dominant polycystic kidney disease (ADPKD) cases [1, 2], is a member of a class of proteins novel in both size and combination of different functional groups [3, 4]. Based on PKD1 gene sequence, polycystin should contain 4302 amino acids with an estimated mo- lecular weight of approximately 460 kDa. By comparison with known sequences in databases, it should be a trans- membrane glycoprotein with the carboxy and the amino ends inside and outside the cell, respectively. The puta- tive extracellular portion includes many functional do- mains seen in extracellular and cell-surface proteins, suggesting that polycystin is likely involved in cell-cell and cell-matrix interactions [3, 4]. The C-terminal cyto- plasmic tail may interact with the C-terminal of the pro- tein encoded by the PKD2 gene, mutated in the other 15% of ADPKD cases [5], which retains similarities to a voltage-activated Ca 2+ and Na + 1 channel protein [6, 7]; in addition, it contains potential phosphorylation sites [3, 4], thus indicating that polycystin might function through a common signaling pathway that is necessary for tubulogenesis and as a channel or pore-forming pro- tein [6, 7]. Consistent with the systemic nature of ADPKD, PKD1 RNA sequences were found not only in the kid- ney but also in different adult tissues, at various levels [8], and in various human cell lines [2]. These include the K562 myeloid leukemia cells [9] that, when cul- tured in the presence of chemical compounds such as hemin, butyric acid, hydroxyurea or 5-azacytidine, can be induced to express a developmental program of erythropoiesis with accumulation of the embryonic and fetal hemoglobins [10]. We, therefore, used the K562 cell line as an experimental approach (1) to character- ize the polycystin expression and distribution in a non- 1 To whom correspondence and reprint requests should be ad- dressed at Dipartimento di Biochimica e Biologia Molecolare, Uni- versita ´ degli Studi, Via Luigi Borsari 46, 44100, Ferrara, Italy. Fax: 39 532 202723. E-mail: sen@dns.unife.it. 0014-4827/98 $25.00 259 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. EXPERIMENTAL CELL RESEARCH 244, 259 –267 (1998) ARTICLE NO. EX984198