Functional type I PTH/PTHrP receptor in freshly isolated newborn rat keratinocytes: identification by RT-PCR and immunohistochemistry.
The presence of identical or distinct type I parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptors in keratinocytes is still a matter of debate. We studied the expression and functionality of PTHrP receptors in freshly isolated keratinocytes from newborn rat skin. Four overlapping primers, amplifying different regions in the rat PTH receptor, were used for reverse transcriptase-polymerase chain reaction (RT-PCR). The first region corresponded to the N-terminal extracellular region and the first transmembrane domain (S/M1), the second region amplified the connecting intracellular and extracellular loops transmembrane domain (E2/M5), the third spanned the range from the transmembrane to the intracellular domain (M4/T), and the fourth region amplified the C-terminal tail (M6/7/T). The PCR products from the keratinocyte RNA were identical to those from kidney RNA of the same rats. The cloned four transcripts showed 100% of homologies with the cDNA sequence from bone ROS cells. Keratinocytes, freshly isolated or present in situ in the epidermis, recognized an anti-PTH receptor antibody (PTH-II) directed against the receptor extracellular domain. Western blotting showed the same protein patterns in keratinocytes, kidney, and ROS cell extracts. Low doses of PTHrP(1-34) (10(-12)-10(-9) M) increased the cell number studied by [3H]thymidine incorporation and DNA content. Treatment with the PTH/PTHrP receptor antagonist [Asn10, Leu11, D Trp12] PTHrP(7-34) or two different PTH receptor antibodies inhibited the increase in cell proliferation induced by PTHrP(1-34). All these findings indicate that newborn rat epidermis and keratinocytes express functional PTHrP receptors, which are identical to type I PTH/PTHrP receptor and are recognized by PTHrP(1-34).
Errazahi
Amina
A
Centre National de la Recherche Scientifique, CNRS UMR 8104--INSERM U.567 Equipe Endocrinologie, Os et Développement, Hôpital Saint Vincent de Paul, Paris and Jouy-en-Josas, France.
Bouizar
Zhor
Z
Lieberherr
Michele
M
Souil
Evelyne
E
Rizk-Rabin
Marthe
M
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D012333:RNA, Messenger; D044168:Receptor, Parathyroid Hormone, Type 1; C054296:parathyroid hormone-related protein (1-34); D004247:DNA