Laminin Increases Both Levels and Activity of Tyrosine Hydroxylase in Calf Adrenal Chromaffin Cells

We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopaminebeta-hydroxylase, and phenylethanolamine-N-methyltransferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaftin cells exposed to laminin. ECENT in vitro studies have shown that the effects of soluble neurotrophic molecules, such as nerve growth factor (NGF) j and brain-derived neurotrophic factor, on neuronal survival and neurite outgrowth are influenced by the extracellular matrix. A variety of cultured cells produce molecules that, when attached to the culture substrate, promote rapid neurite outgrowth (4, 9, 17, 21, 27). Although these undefined molecules do not directly support neuronal survival, they can potentiate survival in response to NGF (10). Similarly, the basement membrane protein laminin also stimulates neurite outgrowth (6, 26, 38) and potentiates the survival of appropriate target neurons in response to both NGF and brain-derived neurotrophic factor (11, 24). Interactions of laminin with a variety of other cell types have been shown to evoke multiple effects, including changes in morphology due to a cytoskeletal reorganization and promotion of cell growth and differentiation. Additionally, it has been demonstrated that growth of melanoma cells on laminin leads to an increased synthesis of melanin (19). Laminin is a well-characterized glycoprotein of molecular weight ~ 1 x 106, with a cross-shaped structure as revealed by rotary shadowing (13). It has several distinct structural domains that have been analyzed by producing proteolytic frag~ Abbreviations used in this paper: ACHE, acetylcholinesterase, DBH, dopaminebeta-hydroxylase; DDC, aromatic amino acid decarboxylase; KRH, KrebsRinger Hepes buffer; LDH, lactate dehydrogenase; NGF, nerve growth factor; PNMT, phenylethanolamine-N-methyltransferase; TH, tyrosine hydroxylase. ments of the molecule (for reviews see references 43 and 48). These fragments have then been used in various biological assay systems, as well as for affinity purification of fragmentspecific antibodies from laminin antisera (36, 42, 44). It has therefore been possible to correlate some of the structural domains of laminin with its functional properties. For example, a heparin-binding fragment (fragment 3) of the molecule (33) has been implicated in the interaction of laminin with embryonic chick sympathetic neurons to potentiate NG] rmediated survival and neurite outgrowth (11). Fragments corresponding to other domains of laminin are involved in its binding to type IV collagen, tumor cells, and hepatocytes (36, 42, 44). Like sympathetic neurons, adrenal chromaffin cells are derived from the neural crest (22), synthesize and release catecholamines, and have NGF receptors (32). However, the response of bovine chromaffin cells to NGF depends on developmental stage: At early stages (early fetus), cells respond to NGF with process formation as well as selective increases in levels of the regulatory enzymes in catecholamine biosynthesis, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT). At later developmental stages (late fetus, calf), only increased enzyme levels are seen in response to NGF treatment. In adult bovine chromaffin cells, despite the presence of receptors, no response has, as yet, been found to NGF treatment (32). In contrast to sympathetic neurons, however, © The Rockefeller University Press, 0021-9525/86/01/0151/09 $1.00 The Journal of Cell Biology, Volume 102, January 1986 151-159 151 on July 4, 2017 jcb.rress.org D ow nladed fom bovine chromaffin cells are not dependent upon NGF for survival at any developmental stage (32). It is therefore of interest to determine whether laminin would potentiate the effect(s) of NGF in bovine adrenal chromaffin cells as in sympathetic neurons. To this end, we chose to work with calf chromaffin cells to address the following questions: (a) Could substrate-bound laminin enable cells to respond to NGF as they had at earlier stages in development, i.e., with process formation as well as with increased enzyme levels? (b) Would laminin increase the potency and/or efficacy of NGF to increase enzyme levels? (c) Which of the functional domains of laminin (neuron vs. non-neuronal cell binding domain) would be effective in interacting with chromaffin cells? The results show that while laminin does not potentiate the NGFmediated increase in enzyme levels, it has a direct effect on TH activity by itself, bringing about both activation and increased amounts of the enzyme. Materials and Methods