It is known that the extracellular matrix regulates normal cell proliferation, and it is assumed that anchorage-independent malignant cells escape this regulatory function. Here we demonstrate that human M24met melanoma cells remain responsive to growth regulatory signals that result from contact with type I collagen and that the effect on proliferation depends on the physical structure of the collagen. On polymerized fibrillar collagen, M24met cells are growth arrested at the G 1͞S checkpoint and maintain high levels of p27 KIP1 mRNA and protein. In contrast, on nonfibrillar (denatured) collagen, the cells enter the cell cycle, and p27 KIP1 is downregulated. These growth regulatory effects involve contact between type I collagen and the collagen-binding integrin ␣2␤1, which appears restricted in the presence of fibrillar collagen. Thus melanoma cells remain sensitive to negative growth regulatory signals originating from fibrillar collagen, and the proteolytic degradation of fibrils is a mechanism allowing tumor cells to escape these restrictive signals.T he extracellular matrix (ECM) is a complex structure of collagens, glycoproteins, elastin, and proteoglycans that, in addition to providing a scaffold for tissues, regulates many fundamental cellular processes such as proliferation, survival, migration, and differentiation (1, 2). It is now appreciated that the ECM provides a complex combination of insoluble signals that, in concert with cell-cell contacts and soluble signals provided by growth factors, affect gene expression and influence cell functions (3-5). It is known that normal cells need contact with the ECM to progress into the cell cycle, and loss of this requirement is a hallmark of malignant cells (6). The mechanisms by which the ECM regulates the growth of anchoragedependent cells have received renewed attention over the past years and have been the subject of investigations that have pointed to cell cycle regulatory proteins that are influenced by contact between cells and the ECM (7-9). On binding to specific cell-surface receptors such as integrins, the ECM exerts its control on cell proliferation by modulating the expression of G 1 regulators in a growth factor-dependent and independent manner. In most conditions, there is cooperative action between growth factors and ECM that results in nonredundant signals that transcriptionally up-regulate cyclins D and E and downregulate the cyclin-dependent kinase (CDK) inhibitors p21 CIP1 , p27 KIP1 , and p57 KIP2 (10). This cooperative action permits cells to pass through the G 1 restriction point and to complete the cell cycle. However, there are other conditions where the ECM has been shown to send growth inhibitory rather than growth stimulatory signals. These conditions depend not only on the composition of the ECM but also on its physical state. Molecules of type I collagen in vivo are organized into striated fibrils. These structures, which are responsible for the resistance of many tissues, are lost on proteolytic degradation of the collagen. ...

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is a key determinant in the homeostasis of the extracellular matrix. We have identified two cis-acting elements involved in the transcriptional regulation of TIMP-2. The first is an inverted CCAAT box located at position ؊73 to ؊69 in the TIMP-2 promoter that binds the transcription factor NF-Y. The second is a GAGGAGGGGG motif located at position ؊107 to ؊98, that binds the transcription factors Sp1 and Sp3. NF-Y and Sp1 cooperate for the basal transcription activity of the promoter. We then determined that TIMP-2 is transcriptionally up-regulated by cAMP analogs. Up-regulation of TIMP-2 by dibutyryl cAMP is a delayed response that requires de novo protein synthesis and does not affect RNA stability. The NF-Y and the Sp1 binding site are both involved in cAMPdependent up-regulation of TIMP-2. Whereas NF-Y is essential for cAMP mediated regulation, Sp1 alone is not sufficient but enhances the activity of NF-Y. Dibutyryl cAMP has no effect on the expression of MMP-2 and MMP-9 and switches the MMP-TIMP balance in favor of the inhibitor.Tissue inhibitors of metalloproteinases (TIMPs) 1 are natural inhibitors of matrix metalloproteinases (MMPs), a family of proteases that degrade proteins of the extracellular matrix (ECM) (1). These inhibitors play an important role in the control of the homeostasis of the ECM under conditions that are associated with intense connective tissue remodeling such as implantation, embryogenesis, organogenesis, wound repair, arthritis, and cancer invasion (2, 3). Four members of the TIMP family, designated TIMP-1 to TIMP-4, have been identified so far in many species including human (4 -8). These inhibitors differ in many aspects such as tissue-specific expression, solubility, and regulation but share a common antiprotease function because of their ability to form stable enzyme-inhibitor complexes with all members of the MMP family. The balance between MMPs and TIMPs is a key element that controls the turnover of the ECM and is itself under the influence of a large variety of growth factors and cytokines that transcriptionally regulate MMP and TIMP expression. Whereas some of these factors such as transforming growth factor ␤ have a coordinated effect that decreases ECM degradation by up-regulating TIMP and down-regulating MMP expression (9, 10), other factors such as interleukin-1, interleukin-6, or oncostatin M have a much less predictable effect on the ECM because they simultaneously up-or down-regulate MMP and TIMP expression (11)(12)(13)(14).The molecular basis for the transcriptional regulation of MMP and TIMP expression has been investigated, and several cis-acting elements in the promoter of many MMPs and TIMPs responsible for transcription control have been identified. In particular, a PEA-3 motif and an AP-1 motif located in close proximity in the promoter of many MMPs and TIMPs have been shown to mediate their enhanced expression by phorbol esters and serum factors (15), and a transform...

The extracellular matrix is a crucial component in determining cell fate. Fibrillar collagen in its native form inhibits cell proliferation, whereas in its monomeric form it stimulates proliferation. The observation of elevated levels of p27 KIP1 in cells plated in the presence of fibrillar collagen has led to the assumption that this kinase inhibitor was responsible for cell cycle arrest on fibrillar collagen. Here we provide evidence that p15INK4b , rather than p27 KIP1 , is the cyclin-dependent kinase inhibitor responsible for G 0 /G 1 arrest of human melanoma cells grown on fibrillar collagen. Additionally, we demonstrate that fibrillar collagen can also arrest cells at the G 2 phase, which is mediated in part by p21 CIP1 . Our data, in addition to identifying cyclin-dependent kinase inhibitors important in cell cycle arrest mediated by fibrillar collagen, demonstrate the complexity of cell cycle regulation and indicate that modulating a single cyclindependent kinase inhibitor does not disrupt cell proliferation in the presence of fibrillar collagen.