Cathepsin L is a lysosomal cysteine proteinase with a major role in intracellular protein catabolism. It also shows the most
potent collagenolytic and elastinolytic activity in vitro of any of the cathepsins. Joseph et al. (1988) presented the complete nucleotide and predicted amino acid
sequence for human preprocathepsin L, demonstrated cathepsin L mRNA in a human tumor, and showed evidence for a higher
molecular weight human kidney transcript.
General function
Enzyme, Hydrolase, Peptidase/Protease
Comment
The lysosomal cysteine protease cathepsin L is synthesized in large amounts and
secreted by many malignantly transformed cells in culture. The secreted protease is potent in degrading collagen, laminin,
elastin, and other structural proteins of basement membranes. To determine whether human cancers synthesize cathepsin L,
the expression of cathepsin L in approximately 100 human tumor samples was determined by quantitative RNA slot blot
analysis using a specific human cathepsin L complementary DNA probe. Results by Chauhan SS et al suggest that cancers in
general express higher levels of cathepsin L than do normal tissues. Kidney and testicular tumors expressed the highest levels
of cathepsin L; non-small cell carcinomas of the lung expressed the next highest levels; and most cancers of the breast, ovary,
colon, adrenal, bladder, prostate, and thyroid expressed elevated levels as well. Cathepsin L may prove useful as a
diagnostic or prognostic marker of human malignancy.
Millest AJ, et al reported effects of an inhibitor of cathepsin L on bone resorption in thyroparathyroidectomized and ovariectomized rats.
Cellular localization
Secreted
Comment
Ovarian function
Follicle atresia, Ovulation, Steroid metabolism
Comment
A 70-kilodalton
(kD) protein complex secreted from rat Sertoli cells was isolated by Boujrad N et al . The complex, composed of 28- and 38-kD proteins,
stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine
3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be
responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins
were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L,
respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis.
Expression regulated by
FSH, LH
Comment
Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period. Garc?V et al. OBJECTIVE: To study in?vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. DESIGN: Experimental study. SETTING: University research unit. PATIENT(S): Twenty-five women of reproductive age. INTERVENTION(S): Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in?vitro fertilization. MAIN OUTCOME MEASURE(S): To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. RESULT(S): The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. CONCLUSION(S): The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation.
The progesterone receptor (PR), a nuclear receptor transcription factor, is induced in granulosa cells of
preovulatory follicles in response to the LH surge and has been shown to be essential for ovulation, because mice
lacking PR fail to ovulate and are infertile. Using these mice as a model in which to elucidate PR-regulated genes in the
ovulation process, R. L. Robker et al 2000 show that the matrix metalloproteinases MMP-2 and MMP-9 are not targets of PR during
ovulation. In contrast, two other proteases, ADAMTS-1 (A disintegrin and metalloproteinase with thrombospondin-like
motifs) and cathepsin L (a lysosomal cysteine protease), are transcriptional targets of progesterone receptor (PR) action. ADAMTS-1 is induced
after LH stimulation in granulosa cells of preovulatory follicles and depends on PR. Cathepsin L is induced in granulosa cells of growing follicles by follicle-stimulating hormone, but the highest levels of cathepsin L mRNA occur in
preovulatory follicles in response to LH in a PR-dependent manner. The identification of these regulated proteases in the
ovary, together with their abnormal expression in anovulatory PR knockout mice, suggests that each plays a critical role
in follicular rupture and represents a major advance in our understanding of the proteolytic events that control ovulation.
Sriraman V,et al reported that Cathepsin L gene expression and promoter activation in rodent granulosa cells.
The cysteine protease cathepsin L exhibits hormone regulated expression during the ovulation. In situ hybridization analyses of immature and PMSG-treated mice and rats ovaries showed that cathepsin L expression in granulosa cells of small, growing follicles increased in periovulatory follicles following hCG stimulation. In the rat ovary, cathepsin L was also expressed in follicles with signs of atresia. To determine the molecular mechanisms that mediate the diverse regulation of this gene in granulosa cells, rat cathepsin L promoter-reporter constructs were analyzed by transient transfection assays in rat granulosa cells and electrophoretic mobility shift assays. A construct containing the transcriptional start site and -244 bp of upstream promoter sequence (-244/+33bp) exhibited inducibility by forskolin, the phorbol ester PMA and an additive effect of both. Within this region three functional Sp1 sites, an overlapping Egr-1 site and a CREB site were identified. Single or double mutants of the above mentioned sites did not alter forskolin/PMA inducibility of the promoter. Mutation of all the three Sp1/Sp3 sites (which also mutated the Egr-1 site) reduced the promoter activation. Mutation of the CREB site in the triple Sp1 mutant construct completely blocked the inducibility of the promoter. When these same constructs were transfected into MCF-7 cells or were co-transfected with an Sp1 expression vector in Drosophila SL2 cells, similar results were obtained. Collectively, the data document that three Sp1/Sp3 binding GC-rich regions and a functional CRE constitute an important transcriptional regulatory complex for expression of the cathepsin L gene in rat granulosa cells.
Ovarian localization
Cumulus, Granulosa, Luteal cells
Comment
Hormonal and molecular characterization of follicular fluid, cumulus cells and oocytes from pre-ovulatory follicles in stimulated and unstimulated cycles. de Los Santos MJ et al. BACKGROUNDThe use of ovarian stimulation, to stimulate a multi-follicular response for assisted reproduction treatments, may force the production of oocytes from follicles that do not reach optimal maturation, possibly yielding oocytes that are not fully competent. The present study aimed to define the follicular environment and oocyte competence of unstimulated pre-ovulatory follicles, to compare it with that of similar-sized stimulated follicles. For this purpose, we analyzed the follicular hormonal milieu, the oocyte meiotic spindle, the embryo development and the cumulus cells gene expression (GE) profiles.METHODS AND RESULTSThe study population was divided in two groups: (i) 42 oocyte donors undergoing unstimulated cycles and (ii) 18 oocyte donors undergoing controlled ovarian stimulation cycles (COS). Follicular fluid was analyzed to quantify the concentrations of estradiol (E2), progesterone (P), FSH, LH, testosterone (T) and androstendione (?4). T was higher in the COS group, while ?4, E2 and LH were significantly higher in unstimulated cycles. The cumulus oophorus cells (CC) surrounding the oocyte were removed and their GE profiles were analyzed with microarrays. There were 18 differentially expressed genes in CC: 7 were up-regulated and 11 were down-regulated in the COS cycles. The microarray was validated by qRT-PCR. The analysis of spindle structure revealed no significant differences between the groups, except for the parameter of length which presented differences. The fertilization ability and embryo morphology on Days 2, 3 and 4 did not show any significant differences between groups.CONCLUSIONSThe use of ovarian stimulation induces changes in the follicular fluid and in CC GE that may affect immune processes, meiosis and ovulation pathways. Although these differences do not seem to relate to early-stage embryo morphology, the implications of some of the molecules, especially ALDH1A2, CTSL and ZNF33B at the CC level, deserve to be addressed in future studies.
Park et al investigated the
expression of cathepsin L by Northern blot analysis with radiolabeled cDNA specific for cathepsin L In six normal
tissues, the highest level of cathepsin L was expressed in liver with the descending order of liver > lung > thymus > ovary >
kidney > esophagus.
Changes in mouse granulosa cell gene expression during early luteinization. McRae RS et al. Changes in gene expression during granulosa cell luteinization have been measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with pregnant mare serum gonadotropin (PMSG) or PMSG followed, 48 h later, by human chorionic gonadotropin (hCG). Granulosa cells were collected from preovulatory follicles after PMSG injection or PMSG/hCG injection and SAGE libraries generated from the isolated mRNA. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by hCG and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in steroidogenesis as well as clusters of genes involved in modeling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signaling. The SAGE libraries described here provide a base for functional investigation of the regulation of granulosa cell luteinization.
Follicle stages
Antral, Preovulatory
Comment
Nishida Y et al reported increased cathepsin L levels in serum in some patients with ovarian cancer. Cathepsin L gene expression and promoter activation in rodent granulosa cells.
Sriraman V,et al 2004 . The cysteine protease cathepsin L exhibits hormone-regulated expression during ovulation. In situ hybridization analyses of immature and pregnant mare serum gonadotropin-treated mouse and rat ovaries showed that cathepsin L expression in granulosa cells of small, growing follicles increased in periovulatory follicles after human chorionic gonadotropin stimulation. In the rat ovary, cathepsin L was also expressed in follicles with signs of atresia. To determine the molecular mechanisms that mediate the diverse regulation of this gene in granulosa cells, rat cathepsin L promoter-reporter constructs were analyzed by transient transfection assays in rat granulosa cells and EMSAs. A construct containing the transcriptional start site and -244 bp of upstream promoter sequence (-244/+33 bp) exhibited inducibility by forskolin, the phorbol ester phorbol myristate acetate, and an additive effect of both. Within this region, three functional specificity protein 1 (Sp1) sites, an overlapping early growth response protein-1 site, and a cAMP regulatory element-binding protein site were identified. Single or double mutants of the above-mentioned sites did not alter forskolin/phorbol myristate acetate inducibility of the promoter. Mutation of all three Sp1/specificity protein 3 (Sp3) sites, which also mutated the early growth response protein-1 site, reduced the promoter activation. Mutation of the cAMP regulatory element-binding protein site in the triple Sp1 mutant construct completely blocked the inducibility of the promoter. When these same constructs were transfected into MCF-7 human breast cancer cells or were cotransfected with an Sp1 expression vector in Drosophila SL2 cells, similar results were obtained. Collectively, the data document that three Sp1/specificity protein 3 binding GC-rich regions and a functional cAMP regulatory element constitute an important transcriptional regulatory complex for expression of the cathepsin L gene in rat granulosa cells.