Date of Award

12-1999

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular Immunology, Virology and Inflammation

Supervisor

Carl D. Richards

Abstract

The progression of an inflammatory response is largely dictated by soluble
factors termed cytokines reknown for their redundant and pleiotropic nature in
modulation of both immune and stromal cells. Individual members of the
interleukin-6 (IL-6)-type cytokine family possess both unique and shared biological
activities. These cytokines may participate in tissue remodelling by promoting
reconstruction ofextracellular matrix (ECM) following nonspecific tissue damage by
inflammatory cells. Consistent with this view, these cytokines upregulate expression
of an ECM protease inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) and
thus may alter net enzymatic degradation of ECM. The overall goal of this thesis
is to examine mechanisms by which TIMP-1 is regulated by IL-6-type cytokines,
especially by the cytokine oncostatin M (OSM). The promoter of TIMP-1 has
therefore been studied in detail to address the mechanisms by which OSM (and IL-6
type cytokines) regulate the transcription of the TIMP-1 gene. The approaches
undertaken have included deletion analysis of the TIMP-1 gene nucleotide
sequences proximal to the start of transcription to define DNA sequences
necessary/sufficient for cytokine-induced TIMP-1 promoter activity. In addition, the
binding of nuclear factors to these DNA elements and cytokine-response elements, their expression and involvement in the regulation of TIMP-1 transcription have been explored.

We have identified sequences proximal to the start of TIMP-1 transcription
that are necessary for maximal responsiveness to OSM and IL-6. Deletion analysis
of the proximal TIMP-1 promoter (-95 to +47 TIMP-1 sequences) has identified a
nucleotide sequence within -59 to -53 ofthe murine TIMP-1 promoter that harbours
an AP-1 consensus DNA binding element. This element is necessary for maximal
OSM or IL-6 induced promoter activity of TIMP1-CAT reporter gene constructs
transfected into human hepatoma HepG2 cells. OSM is the most potent stimulus
(approx. 11-fold for OSM, and 4-fold for IL-6) of this response and additional
sequences 3-prime to +1 ofthe TIMP-1 gene are also necessary for maximal OSM
responsiveness.

Electrophoretic mobility shift assays demonstrated two gel-shifted complexes
which bind the TIMP-1 AP-1 site. An AP-1 gel-shifted complex is present in the
absence of cytokine stimulation ("complex 1"), while OSM and not other IL-6-type
cytokines, stimulated the formation of a second AP-1 gel shifted complex.
Nuclear factors binding to TIMP-1 AP-1 complex 1 include junB, junD and fosrelated
antigens. However, unlike complex1, c-fos is present and necessary for the
formation of the OSM-induced TIMP-1 AP-1 complex2. Consistent with this, OSM
is a potent inducerofc-fos protein expression among IL-6-type cytokines. Both the
formation of complex2 and c-fos expression require new protein synthesis. JunB
and junO are constitutively expressed, while the expression of fos-related antigens are induced in response to OSM. In addition, although PMA was also a potent
inducer of c-fos expression, induction of TIMP-1 promoter activity by the
combination of PMA and IL-6 was comparable to IL-6 alone and did not equal the
significantly higher induction by OSM. Within the same cells, OSM and IL-6 equally
induced STATDNA-binding activity. An Ets-consensus site (nucleotides -45 to -40)
flanking the 3-prime end of the AP-1 site is a weak binding site for Ets-related
nuclear factors, and an SP-1 site near +1 (-11 to -6) is a strong binding siteforSP1
nuclear factors and related SP-1 site binding proteins. No STAT nuclear factor
binding to the proximal TIMP-1 promoterwas detected. Taken together, the TIMP1
AP-1 site and c-fos represent a unique target of OSM signalling and activation of
AP-1 complexes (possibly containing c-fos) by OSM as well as sequences
downstream of TIMP-1 +1 contribute to maximal responsiveness of the promoter
to this cytokine among IL-6 family members.

The contribution of c-fos to OSM-induced TIMP-1 expression was further
explored in murine cells. OSM stimulates the expression of c-fos and activates
STATs 1, 3 and 5 DNA-binding activity in murine fibroblasts. As observed in
human cells, OSM but not other IL-6-type cytokines upregulated c-fos expression
which participated in complexes binding the TIMP-1 AP-1 site. OSM was also
unique among IL-6 family members in activating STAT5 DNA-binding activity in
murine fibroblasts. However, in contrast to observations in human HepG2 cells,
deletion analysis of the TIMP-1 promoter showed that the AP-1 site (-59/-53) was
v not necessary for OSM-mediated upregulation of the TIMP-1 proximal promoter
activity over basal levels in murine NIH3T3 fibroblasts and co-transfection of a
dominant-negative of AP-1 had no effect. However, transfection of dominant-negative
STATs1, 3 or5 (especially STAT3) could diminish cytokine-induced TIMP1
promoter activity. In addition, c-Fos was dispensable for OSM-mediated
upregulation ofTIMP-1 mRNA levels as TIMP-1 expression was detected in wildtype
and c-fos knockout murine lung fibroblasts. Consistent with deletion analysis
ofthe TIMP-1 promoter in human cells, dominant-negative AP-1 expression vectors
abrogated OSM-mediated TIMP-1 promoter activity, while Stat-dominant negative
expression vectors did not. Taken together, the examination of murine and human
systems suggests that AP-1 and STAT nuclear factors can contribute to the
regulation of the TIMP-1 promoter.