Transforming growth factor ,B effects on expression of G1 cyclins and cyclin-dependent protein kinases

Transforming growth factor (31 (TGF-f81) is a potent growth-inhibitory polypeptide. The mechanism of TGF-13, inhibition has been related to its ability to prevent the hyperphosphorylation of retinoblastoma protein (pRb). Several lines of evidence have suggested that cell cycle-regulated protein kinases are responsible for the hyperphosphorylation of pRb. We demonstrate here that TGF-fi1 has profound effects on the expression of genes encoding certain Gl cyclins and their associated kinases, which provides one explanation of TGF-p1 effects on pRb hyperphosphorylation. These results also suggest that the growth-inhibitory effects of TGF-813 in many cells are attributable to its effects on the cell cycle apparatus involved in programming Gl transit. Transforming growth factor /8 (TGF-P3) is a multifunctional cytokine which is involved in controlling cell cycle progression, cell differentiation, cell adhesion, and extracellular matrix deposition in a variety of cell lineages (1, 2). Of the three mammalian forms ofthis protein (TGF-,31, -/32, and -/33), TGF-31 has been studied most extensively over the past decade. One of the readily measured biological activities of TGF-f31 is its antiproliferative action on a variety of cell types. For example, TGF-,31 arrests epithelial cells in the Gl phase of their cell cycle (3, 4). However, the mechanism of growth inhibition of TGF-/31 remains unclear. Several cellular genes that are important in the control of cell proliferation have been shown to be affected by TGF-/31 treatment-e.g., the transcription of the c-myc gene is inhibited by TGF-31 (5). The negative growth effects of TGF-/31 have been linked to a tumor suppressor gene product, the retinoblastoma protein, pRb. Specifically, it has been shown that upon TGF-,31 treatment, the hyperphosphorylation of pRb is prevented, pRb remains hypophosphorylated, and cells become arrested in late Gl (3). Since the hyperphosphorylation of pRb, which normally occurs in late Gl, is thought to represent a molecular switch that converts pRb from an active growth-suppressing form into an inactive form (6-9), it has been hypothesized that TGF-,31 exerts its inhibitory effects on the cell cycle progression by preventing this hyperphosphorylation. Because hyperphosphorylation of pRb appears to be the key regulator of its function, the identity of the responsible kinases has attracted much interest. Tryptic mapping of the sites of phosphorylation on pRb in vivo suggested that pRb is phosphorylated by a subset of cdc2-related kinases, termed cyclin-dependent protein kinases (cdks) (10, 11). Recently a number of known or putative cdks have been described (12, 13). The activity and substrate specificity ofcdks are thought to be regulated in turn by association with their regulatory subunits, the cyclins. Five classes ofmammalian cyclins have The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. been enumerated to date: A, B (including Bi and B2), C, D (including Dl, D2, and D3), and E (14-17). These various cyclins have been assigned roles in specific phases of the cell cycle. Thus, cyclin B in association with cdc2 has been shown to promote G2/M transition (18). The expression of cyclin A occurs at the onset of S phase, and this cyclin associates with both cdc2 and cdk2 (12, 19, 20). Cyclins C, D, and E are defined as Gl cyclins, as their expression occurs mainly in Gl. The expression of cyclin E occurs with sudden onset in mid/late Gl in parallel with the appearance of cyclin E'cdk2 complexes having strong kinase activity (15, 20). In contrast, cyclins C and D showed rather constant expression through a cell cycle (15). It has been recently found that cyclin Dl can form a complex with cdk4 (21); however, no kinase activity has yet been found to associate with cyclin C. The timing of initial pRb hyperphosphorylation in late Gl coincides closely with the appearance of cyclin E mRNA. For this reason, it has been speculated that cyclin E is responsible for the initial hyperphosphorylation of pRb, while cyclin A is responsible for subsequent maintenance of pRb hyperphosphorylation through S phase and into G2. To understand the mechanism of action of TGF-f31 in regulating cell cycle progression, we have further explored its effects on the actions of cyclins in the Gl phase of the cell cycle. As described here, TGF-/31 has profound effects on the expression of genes encoding Gl cyclins and their associated cdks. These effects provide important insights into its mechanism of cell growth regulation. MATERIALS AND METHODS Cell Culture. Human keratinocytes [HaCaT cells (22), a gift from P. Boukamp] were cultured in a minimal essential medium (aMEM, GIBCO) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) in a 5% CO2 incubator at 37°C. Cell Synchronization and TGF-,81 Treatment. Cells were plated at the density of 1-2 x 106 cells per 10-cm2 dish and grown in aMEM containing 10% FBS for 36-48 hr until the cell monolayer became confluent. The cells were then cultured in aMEM in the absence of serum for 3 days to achieve quiescence. The cells were restimulated with fresh medium containing 10% FBS. Bovine TGF-/31 (kindly provided by P. Segarini, Celtrix) was added to a final concentration of 2 ng/ml at the beginning of the serum addition or at 2-hr intervals after serum stimulation. DNA Synthesis Measurement. DNA synthesis of the synchronized cells was determined by detection of 5-bromodeoxyuridine (BrdUrd) incorporation into DNA. The cells were grown on glass coverslips and BrdUrd was added with serum. Abbreviations: TGF-pi, transforming growth factor Pj; cdks, cyclindependent protein kinases; pRb, retinoblastoma protein; FBS, fetal bovine serum; BrdUrd, 5-bromodeoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.