Temporal and spatial regulation of gene expression mediated by the promoter for the human tissue inhibitor of metalloproteinases‐3 (TIMP‐3)‐encoding gene

Abstract

A complex interplay between enzymes involved in extracellular matrix formation and their inhibitors is thought to control organogenesis during mammalian development. Disturbance of this balance may result in a wide range of diseases, including macular degeneration, arthritis, and tumor metastases. In order to define elements which may be involved in regulating human tissue inhibitor of metalloproteinase 3 (TIMP3) expression, we isolated and sequenced a clone containing 1315 bp of the 5'-upstream region of the human TIMP-3-encoding gene. A 1.2 kb fragment of this clone, which contains multiple motifs which are binding sites for known transcription factors, was used to drive expression of the lacZ reporter gene in multiple lines of transgenic mice. TIMP3 promoter activity, detected through beta-galactosidase histochemical assay, was observed at high levels in selected tissues, the identity of which varied according to developmental stage. TIMP3 promoter activity was detected at embryonic and early postnatal stages in tissues undergoing extensive remodeling, such as developing somites, bones and joints, choroid plexus, webs between the digits, and the spongiotrophoblastic portion of the placenta. In adulthood, TIMP3 promoter activity was restricted to a few tissues which exhibit high metabolic activity or rapid turnover. These include the retinal pigment epithelium (RPE), cells of the kidney cortex, hair follicles, gingiva, ovarian follicles, and testis. The results suggest that TIMP3 expression plays an active role in developmental patterning and in the maintenance of specific differentiated tissues.

TGF‐β‐induced expression of tissue inhibitor of metalloproteinases‐3 gene in chondrocytes is mediated by extracellular signal‐regulated kinase pathway and Sp1 transcription factor

“…To further investigate the role of Sp1 transcription factor in TIMP‐3 regulation, human SW1353 cells were transiently cotransfected with the constant amounts of human TIMP‐3 promoter‐firefly luciferase vector (5′‐flanking region from −940 to +376, Zeng et al, 1998), pRL‐CMV, an internal control plasmid expressing Renilla luciferase, different amounts of CMV‐Sp1 (expressing Sp1 under the CMV promoter) and mock (pGL3 basic) DNA to keep equal amount of DNA. As shown in Figure 8, increasing amounts of Sp1 significantly enhanced TIMP‐3 promoter‐driven luciferase activity compared to basal levels.…”

“…Cells were detached by trypsinization, trypsin removed by centrifugation and suspension incubated with ODN‐calcium phosphate precipitate for 10 min and plated in serum containing medium for 3 h. Medium was removed, cells washed with PBS, allowed them to recover for 16 h, maintained in serum‐free medium for 36 h and then stimulated with TGF‐β for 24 h. Equal amount (20 μg) of protein was analyzed for TIMP‐3 protein levels as above. In other experiments, 2 μg of TIMP‐3 promoter luciferase (−940 to +376 region; Zeng et al, 1998), cytomegalovirus (CMV)‐Renilla luciferase (0.2 μg, transfection control), CMV‐Sp1 expression vector (0–1.2 μg) (from Dr. Stephen Smale, UCLA), and different amounts of mock DNA (pGL3 basic, Promega) were cotransfected by the modified calcium phosphate procedure described above and luciferase activity analyzed with Promega Dual‐Luciferase Reporter assay System and Turner Designs Luminometer TD‐20/20 according to their recommended procedures.…”

Altered microRNA expression in patients with non-obstructive azoospermia

“…For example, three possible target genes for down-regulated miRNAs, TIMP3, SOX9 and GADD45G , were significantly increased in infertile testis [ 24 ]. TIMP-3, a potential target of four down-regulated miRNAs (miR-1, miR-181a, miR-221 and miR-9*), is thought to play an active role in testicular development and differentiation [ 30 ]. The transcription factor SOX9, a putative target of miR-145, is required for Sertoli cell maturation and normal spermatogenesis [ 31 ].…”

Matrix metalloproteinases and their inhibitors regulate in vitro ureteric bud branching morphogenesis

“…Matrix metalloproteinases (MMPs) and their inhibitors have been implicated in these complex processes (5-7, 10, 16, 29). Several MMPs and tissue inhibitors of metalloproteinases (TIMPs) have been shown to be widely expressed during development (1,4,21,30,34). In the developing kidney, involvement of MMPs and TIMPs has been demonstrated in organ culture.…”