Subclones of a rat parathyroid cell line (PT-r): Regulation of growth and production of parathyroid hormone-related peptide (PTHRP)

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Abstract
Four subclones from a rat parathyroid cell line (PT-r cell) have been isolated, and morphological and functional characteristics have been examined. Subclones 1 and 2 display a polygonal shape, show growth and secretory responses to calcium (half-maximal suppressions at 1.2 and 1.7 mM, respectively), and respond to secretin with cAMP production (14.5-fold and 16.9-fold over basal) and hormone secretion (41 and 58% over basal). Subclone 4 is elongated in form and does not respond to calcium or secretin. Subclone 3 shows mixed morphology, elongated and polygonal shapes, with moderate response to calcium (half-maximal suppression at 1.7 mM) and secretin (cAMP, 3.2-fold increase and hormone secretion, 50% increase over basal). The clones were tested for content of messenger RNA (mRNA) representing parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP). Only PTHRP mRNA was found. The peptide released is virtually all PTHRP. PTH mRNA was not detected even with a sensitive RNA probe. The amount of mRNA for PTHRP closely paralleled the amount of PTH-like bioactivity released into the medium from each clone (144.7 ± 12.1, 110.0 ± 12.9, 68.0 ± 5.6, and 39.9 ± 2.4 pgEq of rat PTH-(-34) per 107 cells per 12 h in a medium with 0.7 mM ionized calcium, from subclones 1, 2, 3, and 4, respectively). Culture conditions, low-density passage (less than 1:50 split ratio) or high-density passage (greater than 1:10 split ratio), affected morphology and function of the clones 1 and 2. They became elongated and functionally dedifferentiated like subclone 4 after 3 months of high-density culture. Upon dedifferentiation, morphological and functional properties were not reversible with high calcium, 1,25-(OH)2D3, or retinoic acid. These clones may be useful for studies of phenotypic expression, calcium regulation, and gene expression and interrelationships among these functions.