Molecular Cloning and Characterization of Human Tissue Inhibitor of Metalloproteinase 4

Abstract

The tissue inhibitors of metalloproteinases (TIMPs) constitute a family of proteins, of which three members have so far been described. Using the expressed sequence tag sequencing approach, we have identified a novel TIMP-related cDNA fragment and subsequently cloned a fourth human TIMP (TIMP-4) from a human heart cDNA library. The open reading frame encodes a 224-amino acid precursor including a 29-residue secretion signal. The predicted structure of the new protein shares 37% sequence identity with TIMP-1 and 51% identity with TIMP-2 and -3. The protein has a predicted isoelectric point of 7.34. The open reading frame-directed expression of TIMP-4 protein in MDA-MB-435 human breast cancer cells showed metalloproteinase inhibitory activity on reverse zymography. By Northern analysis, only the adult heart showed abundant TIMP-4 transcripts with a 1.4-kilobase predominant transcript band; very low levels of the transcripts were detected in the kidney, placenta, colon, and testes, and no transcripts were detected in the liver, brain, lung, thymus, and spleen. This unique expression pattern suggests that TIMP-4 may function in a tissue-specific fashion in extracellular matrix homeostasis. Matrix metalloproteinases (MMPs)1 play a critical role in ECM homeostasis. Controlled remodeling of the ECM is an essential aspect in the process of normal development, and deregulated remodeling has been indicated to have a role in the etiology of diseases such as arthritis, periodontal disease, and cancer metastasis (1-5). The overproduction and unrestrained activity of MMPs has been linked to malignant conversion of tumor cells (4 -12). The down-regulation of MMPs may occur at the levels of transcriptional regulation of the genes and activation of secreted proenzymes and through interaction with specific inhibitor proteins such as TIMPs. TIMPs are secreted multifunctional proteins that play pivotal roles in the regulation of ECM metabolism. Their most widely recognized action is as inhibitors of matrix MMPs. Thus, the net MMP activity in the ECM is the result of the balance between activated enzyme levels and TIMPs levels. Augmented MMP activity is associated with the metastatic phenotype of carcinomas, especially breast cancer (7-9, 13-16); the decreased production of TIMP could also result in greater effective enzyme activity and invasive potentials (17-19). These results suggest that an increase in the amount of TIMPs relative to MMPs could function to block tumor cell invasion and metastasis. In fact, tumor cell invasion and metastasis can be inhibited by up-regulation of TIMP expression or by an exogenous supply of TIMPs (17, 40 -44).Three mammalian TIMPs have been characterized at the sequence level: TIMP-1 (20), TIMP-2 (21), and 36). The proteins are classified based on structural similarity to each other as well as their ability to inhibit matrix metalloproteinases. There have been other reports of inhibitors of metalloproteases (IMPs) with characteristics different from these known TIMPs. In some cases these activitie...

Coordinated peak expression of MMP-26 and TIMP-4 in preinvasive human prostate tumor

“…Four members of the human TIMP family have been identified as follows: TIMP-1, TIMP-2, TIMP-3, and TIMP-4 [14][15][16][17]. The cleavage of synthetic peptides in vitro by MMP-26 is inhibited by TIMP-1, TIMP-2, and TIMP-4, with TIMP-4 displaying the greatest inhibitory potency [7,13].…”

“…TIMP-4 is a tight-binding and slow-binding inhibitor of MMP-26, with an apparent K i value of 0.62 nM [13]. TIMP-4 mRNA has been detected in a variety of normal tissues, including those of the heart, kidney, pancreas, colon, testis, endometrium, and placenta [17][18][19]. Under normal conditions, MMPs and TIMPs are expressed at low levels in most adult tissues, but may become upregulated in pathophysiologic conditions such as wound healing and tumor progression.…”

Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas

“…The TIMP family is comprised of 4 gene products (TIMPs 1-4) which inhibit secreted MMPs with roughly comparable potencies (Apte et al, 1995;Greene et al, 1996;Blavier et al, 1999). Individual TIMPs differ markedly in their pro-MMP interactions and gene regulatory mechanisms.…”

A phase II trial of marimastat in advanced pancreatic cancer

“…Collectively, they are able to degrade all components of the extracellular matrix (Jones et al, 1999). MMP activity is regulated by four specific tissue inhibitors of the metalloproteinases (TIMPs) (Greene et al, 1996;Sellers et al, 1997;Stetler-Stevenson, 1989;Uria et al, 1994). MMPs have an important physiological role in embryonic development (Brenner et al, 1989), bone remodelling (Delaisse and Vaes, 1992) and wound healing (Wolf et al, 1992), and a major role in pathological processes including rheumatoid arthritis (Harris, 1990) and cancer invasion and metastasis (Liotta and Stetler-Stevenson, 1991).…”