Protein Kinase A Is a Negative Regulator of Renal Branching Morphogenesis and Modulates Inhibitory and Stimulatory Bone Morphogenetic Proteins

doi:10.1074/jbc.274.37.26305

Protein Kinase A Is a Negative Regulator of Renal Branching Morphogenesis and Modulates Inhibitory and Stimulatory Bone Morphogenetic Proteins*

From the ^‡Division of Nephrology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, the ^§Program in Developmental Biology, The Hospital for Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, and the ^¶Division of Nephrology, The Toronto Hospital, University of Toronto, Toronto, Ontario M5G 1X8, Canada

Abstract

Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961–F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.

Footnotes

↵* This work was supported by grants from The Hospital for Sick Children Research Training Center (to I. R. G. and S. G.), the Hospital for Sick Children Clinician-Scientist Training Program, the Molly Towell Perinatal Research Foundation and the Kidney Foundation of Canada Fellowship Program (to T. D. P.), the Medical Research Council of Canada (to C. W.), the Medical Research Council of Canada Fellowship Program (to M. M.-S.), the National Cancer Institute of Canada, Terry Fox Fund and Medical Research Council of Canada (to J. L. W.), and the Medical Research Council of Canada and Kidney Foundation of Canada (to N. D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‖ To whom correspondence should be addressed: Division of Nephrology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5667; Fax: 416-813-6271; E-mail: norman.rosenblum@sickkids.on.ca.
↵2 I. R. Gupta, M. Macias-Silva, S. Kim, X. Zhou, C. Whiteside, J. L. Wrana, N. D. Rosenblum, submitted for publication.
Abbreviations:

BMP

bone morphogenetic protein

PKA

protein kinase A

HGF

hepatocyte growth factor

8-Br-cAMP

8-bromo-cAMP

DIC

differential interference contrast

TUNEL

terminal deoxynucleotidyl transferase-mediated labeling

FBS

fetal bovine serum

DMEM-F12

Dulbecco’s modified Eagle’s medium-Ham’s F-12 nutrient mixture

CREB

cAMP-response element-binding protein

TGF

transforming growth factor

PBS

phosphate-buffered saline

mIMCD

mouse inner medullary collecting duct

PKI

protein kinase A inhibitor

DAB

diaminobenzidine tetrahydrochloride
- Received February 8, 1999.
- Revision received June 1, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.