Expression and Signaling of Parathyroid Hormone-Related Protein in Cultured Podocytes

Abstract

Podocyte function appears to be regulated by vasoactive factors. In vivo podocytes express parathyroid hormone-related protein (PTHrP), the N-terminal fragment of which has vasoactive properties. Since the signaling pathway(s) of PTHrP(1–36) are unknown in podocytes, differentiated cells of a conditionally immortalized mouse podocyte cell line were studied. Gene expression of PTHrP and the PTH/PTHrP receptor was investigated by RT-PCR; protein distribution of PTHrP was examined by immunofluorescence. Accumulation of cAMP was determined by an enzyme immunoassay; [Ca²⁺]_i was measured by fura-2 ratio imaging. PTHrP and PTH/PTHrP receptor mRNA was detected in differentiated podocytes. Immunoreactive PTHrP exhibited a granular distribution in the cytoplasm of differentiated podocytes. With regard to the signaling pathway(s) of PTHrP(1–36), a concentration-dependent increase of cAMP levels with an EC₅₀ value of 4 ± 2 nM was found. PTHrP(1–36) (1 µM) increased cAMP levels 5.5 ± 1.1-fold above baseline as compared with a 25.4 ± 4.2-fold increase in response to forskolin (10 µM). The PTH/PTHrP receptor antagonist PTHrP(7–34) significantly diminished the PTHrP(1–36)-induced cAMP increase. While superfusion of podocytes with bradykinin (100 nM) increased [Ca²⁺]_i, PTHrP(1–36) (100 nM) was without effect on [Ca²⁺]_i. However, PTHrP(1–36) attenuated the bradykinin-induced increase in [Ca²⁺]_i. Our results suggest that PTHrP is an autocrine hormone in podocytes, which selectively activates the cAMP pathway through the PTH/PTHrP receptor.

cAMP pathway in podocytes

“…Treatment of podocytes with cAMP stimulates aminopeptidase A activity (Stefanovic et al, 1992), an enzyme that participates in the breakdown of angiotensin II (Wolf et al, 1997). We have shown that stimulation of podocytes with PTHrP decreases the accumulation of [Ca 2ϩ ] i in response to bradykinin (Endlich et al, 2001b). cAMP enhances the cGMP formation in podocytes (Ledoux et al, 1997).…”

“…Thus, prostaglandin and PTH/PTHrP receptors on podocytes are stimulated in a paracrine/autocrine fashion. Whereas prostaglandin E 2 increases not only cAMP levels via the EP 4 receptor but also [Ca 2ϩ ] i via the EP 1 receptor in podocytes (Bek et al, 1999b), PTHrP is a pure activator of the cAMP pathway in podocytes (Endlich et al, 2001b). Thus, PTHrP might possess a specific role as a podocyte hormone.…”

Growth Hormone (GH)-dependent Expression of a Natural Antisense Transcript Induces Zinc Finger E-box-binding Homeobox 2 (ZEB2) in the Glomerular Podocyte

“…Cell Culture-Conditionally immortalized mouse podocyte cells (MPC-5) (17) were grown according to a protocol detailed previously (18). Briefly, cells were cultured under growth-permissive conditions on rat tail collagen type I-coated plastic dishes (BD Biosciences) at 33°C with 100% relative humidity and 5% CO 2 in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin, 100 g/ml streptomycin (Invitrogen), and 10 units/ml mouse recombinant ␥-interferon (Sigma).…”

Cell Biology of the Glomerular Podocyte

“…PTH-related protein is also expressed in podocytes (438). Differentiated mouse podocytes possess mRNA of the PTH-related peptide (PTHrP) and PTH/PTHrP receptors (108 (108). Activation of PKA by cAMP may, as in other cell types, reduce contractility and modulate the assembly of actin filaments by phosphorylation of myosin light-chain kinase (155).…”