Molecular Endocrinology, Vol 8, 139-147, Copyright © 1994 by Endocrine Society

ARTICLES

Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation

SM Kaiser, M Sebag, JS Rhim, R Kremer and D Goltzman
Department of Medicine, McGill University, Montreal, Quebec, Canada.

We have used antisense RNA technology to inhibit endogenous PTH-related peptide (PTHRP) production in an established human keratinocyte cell line, HPK1A, to assess the role of PTHRP as a modulator of cell differentiation. Initially to determine the specificity of any alterations in cell function that might be observed, HPK1A cells and Rat-2 fibroblasts (which do not synthesize PTHRP) were both infected with the same retrovirus (pYN) containing antisense PTHRP. In contrast to antisense-infected HPK1A cells (HPK1A-AS), which show accelerated growth indices when endogenous PTHRP production is blocked, antisense- infected Rat-2 cells (Rat-2-AS) displayed no increase in cell proliferation. Consequently, this alteration in HPK1A cell function appeared to be specific to the inhibition of PTHRP production. In HPK1A- AS cells, no PTHRP transcript was observed in cytoplasmic RNA, and none was sequestered in a nuclear RNA preparation. Therefore, hybridization with the antisense strand appears to destabilize PTHRP mRNA, leading to rapid disappearance of the sense-antisense heteroduplex. We then examined the effect of PTHRP inhibition on keratinocyte differentiation using three indices. PTHRP inhibition in HPK1A-AS cells resulted in reduced high mol wt keratin production, as assessed by immunocytochemistry. Expression of mRNA encoding transglutaminase and involucrin was decreased in HPK1A-AS cells compared to that in control cells under conditions of high ambient calcium. Involucrin protein levels were also diminished in HPK1A-AS cells in parallel with the reduced levels of involucrin gene expression. These data, therefore, show that interference with PTHRP production inhibits expression of maturation-specific keratinocyte indices and indicate that endogenous PTHRP acts to enhance differentiation in this keratinocyte model.

This article has been cited by other articles:

				S. A. Rabbani, P. Khalili, A. Arakelian, H. Pizzi, G. Chen, and D. Goltzman Regulation of Parathyroid Hormone-Related Peptide by Estradiol: Effect on Tumor Growth and Metastasis in Vitro and in Vivo Endocrinology, July 1, 2005; 146(7): 2885 - 2894. [Abstract] [Full Text] [PDF]

				M. H. C. Lam, R. J. Thomas, K. L. Loveland, S. Schilders, M. Gu, T. J. Martin, M. T. Gillespie, and D. A. Jans Nuclear Transport of Parathyroid Hormone (PTH)-Related Protein Is Dependent on Microtubules Mol. Endocrinol., February 1, 2002; 16(2): 390 - 401. [Abstract] [Full Text] [PDF]

				V. A. Tovar Sepulveda, X. Shen, and M. Falzon Intracrine PTHrP Protects against Serum Starvation-Induced Apoptosis and Regulates the Cell Cycle in MCF-7 Breast Cancer Cells Endocrinology, February 1, 2002; 143(2): 596 - 606. [Abstract] [Full Text] [PDF]

				L. Carpio, J. Gladu, D. Goltzman, and S. A. Rabbani Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E489 - E499. [Abstract] [Full Text] [PDF]

				K. El Abdaimi, V. Papavasiliou, D. Goltzman, and R. Kremer Expression and regulation of parathyroid hormone-related peptide in normal and malignant melanocytes Am J Physiol Cell Physiol, October 1, 2000; 279(4): C1230 - C1238. [Abstract] [Full Text] [PDF]

				M. E. Wlodek, K. T. Westcott, P. W. M. Ho, A. Serruto, R. Di Nicolantonio, W. Farrugia, and J. M. Moseley Reduced fetal, placental, and amniotic fluid PTHrP in the growth-restricted spontaneously hypertensive rat Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2000; 279(1): R31 - R38. [Abstract] [Full Text] [PDF]

				M. Falzon and P. Du Enhanced Growth of MCF-7 Breast Cancer Cells Overexpressing Parathyroid Hormone-Related Peptide Endocrinology, May 1, 2000; 141(5): 1882 - 1892. [Abstract] [Full Text] [PDF]

				R. Re The Nature of Intracrine Peptide Hormone Action Hypertension, October 1, 1999; 34(4): 534 - 538. [Abstract] [Full Text] [PDF]

				M. H. C. Lam, L. J. Briggs, W. Hu, T. J. Martin, M. T. Gillespie, and D. A. Jans Importin beta  Recognizes Parathyroid Hormone-related Protein with High Affinity and Mediates Its Nuclear Import in the Absence of Importin alpha J. Biol. Chem., March 12, 1999; 274(11): 7391 - 7398. [Abstract] [Full Text] [PDF]

				K. Meerovitch, S. Wing, and D. Goltzman Proparathyroid Hormone-related Protein Is Associated with the Chaperone Protein BiP and Undergoes Proteasome-mediated Degradation J. Biol. Chem., August 14, 1998; 273(33): 21025 - 21030. [Abstract] [Full Text] [PDF]

				T. Massfelder, P. Dann, T. L. Wu, R. Vasavada, J.-J. Helwig, and A. F. Stewart Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: A critical role for nuclear targeting PNAS, December 9, 1997; 94(25): 13630 - 13635. [Abstract] [Full Text] [PDF]

				K. Meerovitch, S. Wing, and D. Goltzman Preproparathyroid Hormone-related Protein, a Secreted Peptide, Is a Substrate for the Ubiquitin Proteolytic System J. Biol. Chem., March 7, 1997; 272(10): 6706 - 6713. [Abstract] [Full Text] [PDF]

				R. H. Hastings, R. M. Ryan, C. T. D'Angio, B. A. Holm, A. Patel, R. Quintana, E. Biederman, D. W. Burton, and L. J. Deftos Parathyroid hormone-related protein response to hyperoxic lung injury Am J Physiol Lung Cell Mol Physiol, June 1, 2002; 282(6): L1198 - L1208. [Abstract] [Full Text] [PDF]